Parenteral formulations of lipophilic pharmaceutical agents and methods for preparing and using the same

ABSTRACT

There may be provided compositions of lipophilic pharmaceutical agents with improved solubility and stability. For example, there may be provided a non-aqueous composition that comprises a lipophilic pharmaceutical agent, and an amphiphilic polymeric solvent such as PEG400 but essentially free of organic solvents and non-solubilized particles. The composition may be further diluted with a desired aqueous diluent such as an infusion fluid for parenteral administration to a subject such as a human. The compositions may be useful for the treatment for diseases or conditions that are sensitive to lipophilic agents, such as infectious diseases, malignant or autoimmune diseases.

This application is a continuation of U.S. application Ser. No.16/014,908, filed Jun. 21, 2018, which is a continuation of U.S.application Ser. No. 15/451,748, filed Mar. 7, 2017, now U.S. Pat. No.10,028,949, which is a divisional of U.S. application Ser. No.15/154,919, filed May 13, 2016, now U.S. Pat. No. 9,724,345, which is acontinuation of U.S. application Ser. No. 13/452,033, filed Apr. 20,2012, now U.S. Pat. No. 9,364,433, which claims priority to U.S.Provisional Application No. 61/480,259, filed Apr. 28, 2011, each ofwhich is hereby incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is related generally to an improved compositionand method for preparing parenteral formulations comprising solubilizedlipophilic pharmaceutical agents and using these formulations intreatment of diseases such as malignant and autoimmune diseases,infectious disorders, or for use in conditioning therapy precedinghematopoietic stem cell transplantation.

Description of Related Art

Lipophilic drug substances having low water solubility are a growingclass of drugs with increasing applicability in a variety of therapeuticareas for a variety of pathologies. Many compounds approved forpharmaceutical use are lipophilic compounds with limited solubility andbioavailability. Relatively insoluble compounds, i.e., solubility inwater of less than 200 μg/mL may show promising pharmaceutical activity,but their development as pharmaceuticals, particularly in parenteraldosage form, present a significant challenge to the pharmaceuticalindustry. Among the main barriers for effective drug delivery aresolubility and stability. To be absorbed in the human body, a compoundhas to be soluble in both water and fats (lipids). However, solubilityin water is often associated with poor fat solubility and vice-versa.

Solubility and stability are, therefore, major obstacles hindering thedevelopment of therapeutic agents. Aqueous solubility is a necessary butfrequently elusive property for formulations of the complex organicstructures found in pharmaceuticals. Traditional formulation systems forvery insoluble drugs have involved a combination of organic solvents,surfactants and extreme pH conditions. These formulations are oftenirritating to the patient and may cause adverse reactions. At times,these methods are inadequate for solubilizing enough of a quantity of adrug for a parenteral formulation.

Therefore, there exists a need for compositions and methods involvingformulations comprising solubilized and stable lipophilic pharmaceuticalagents, such as busulfan.

As a particular example, the bifunctional DNA-alkylating agent Busulfan(Bu; 1,4-butanediol dimethanesulfonate, aka Butane-1,4-diyldimetanesulfonate; C₆H₁₄O₆S₂) has over the last several decades earnedan impressive reputation for its chemotherapeutic efficacy againstnumerous malignant diseases. This is most readily appreciated, however,in its activity against myeloid neoplasms, such as that exemplified bychronic myelogenous leukemia (CML) (Haddow and Timmis, 1953; Hoffman etal., 1991).

The therapeutic benefit obtained with Bu in single agent (alkylatingagent) therapy in treatment of CML was achieved through its generalmyelotoxicity. It has lately been increasingly replaced by targetedtherapy with tyrosine kinase inhibitors, which may selectivelydown-regulate the aberrant proliferation of the malignant clone(s) andrestore normal polyclonal hematopoiesis.

On the other hand it was recognized by Santos and coworkers, and furtherrefined by Tutschka and coworkers, that the remarkably potent (andselective) myelosuppressive activity of Bu, in addition to itspronounced antileukemic efficacy, makes it an almost ideal agent for usein pretransplant conditioning therapy for patients undergoinghematopoietic stem cell transplantation for malignant-, autoimmune-, orgenetic diseases provided that its myelosuppressive activity was pairedwith the immunosuppressive activity of a second agent, cyclophosphamide(Cy), was usually the preferred “partner” in this setting. Variants ofthis “Bu-Cy” combination quickly became recognized as (an) acceptablealternative(s) to the (at the time) more commonly used combinations oftotal body irradiation (TBI) and Cy (Santos and Tutschka, 1974; Santoset al., 1974; Tutschka et al., 1987; Ciurea et al., 2009). When moreexperience accumulated with the Bu-Cy combinations it became apparentthat the unpredictable intestinal absorption and erratic bioavailabilityof oral Bu was a contributory reason for high peri-transplant morbidityand mortality, most importantly caused by serious liver toxicity ordrug-induced toxic hepatitis, clinically referred to as veno-occlusivedisease of the liver (VOD). The risk of dying of VOD and othertreatment-related complications was reported to be as high as 30-50%already within the first 100 days after the HSCT (Blaise et al., 1992;Devergie et al., 1995; Hartman et al., 1998; Socie et al., 2001; Ciureaet al., 2009). The toxicity from virtually any myeloablative preparativeregimen has been associated with the development of VOD (Jones et al.,1987; McDonald et al., 1993; Bearman, 1995), but VOD and/or hepato-renalfailure after administration of oral Bu (combined with Cy) has commonlybeen considered a “trade-mark” toxicity associated with high-dose Bu(both the original BuCy4 [4 days of Cy] and the variant BuCy2 [2 days ofCy] regimens) (Santos et al., 1983; Tutschka et al., 1987; Grochow etal., 1989; Grochow, 1993; Slattery et al., 1997; Dix et al., 1996).Additionally, oral Bu is associated with a hepatic first-pass extractioneffect that results in locally high Bu concentrations in theportal-hepatic venous system, and this may contribute to the risk forVOD (Peters et al., 1987). However, in addition to Bu, Cy is clearlyhepatotoxic. The findings of McDonald and coworkers suggest thatinter-individual differences in metabolic drug handling are ofimportance for developing VOD, such that, in addition to Bu, Cyconceivably contributes to the overall risk of VOD (McDonald et al.,2003). Thus, the risk of VOD is related to the drug-induced metabolicstress on the liver, especially when both agents depend on hepaticglutathione (GSH) stores and on hepatic Glutathione-S-Transferase (GST)activity for their detoxification (McDonald et al., 2003; Hassan et al.,2000).

In addition to VOD, neurotoxicity was associated with Bu in animals(Deeg et al., 1999). Convulsions in a human receiving oral Bu were firstreported by Marcus and Goldman (1984). The incidence of neurotoxicity,especially serious generalized seizure activity, after Bu-basedconditioning therapy has been estimated to be as high as 10% in adults(Santos, 1989), and approximately 7% in children (Vassal et al., 1990).Vassal et al reported that higher doses (>600 mg/m² or 16 mg/kg) areassociated with an increased probability of neurotoxic manifestations(Vassal et al., 1989). Seizures are more common in older patients, andthey appear to be dose-dependent both in adults and children. Seizuresare related to Bu's limited plasma binding and therefore its ability tocross the blood-brain-barrier (Vassal et al., 1990; Vassal et al., 1989;Hassan et al., 1989; Meloni et al., 1992). In adults, seizures typicallyoccur in the 3^(rd) or 4^(th) day of Bu administration, probably as aresult of tissue drug accumulation (Marcus and Goldman, 1984; Hassan etal., 1989; Meloni et al., 1992; Kobayashi et al., 1998; Martell et al.,1987; Sureda et al., 1989). Even without overt seizure activity EEGabnormalities occur in up to 60% of patients (Kobayashi et al., 1998).These problems necessitate that various anticonvulsant medications beused for seizure prophylaxis (Meloni et al., 1992; Kobayashi et al.,1998; Grigg et al., 1989; Meloni et al., 1995; Chan et al., 2002; Hassanet al., 1993).

The practical limitations in using oral Bu in high-dosepretransplantation conditioning therapy are primarily related to itsunpredictable and erratic bioavailability due to variable intestinalabsorption. Available clinical trial data and concerns related to oralBu toxicity formed the basis for our hypothesis that an IV Buformulation might cause less stress to the liver, since parenteraladministration will yield complete dose assurance with 100%bioavailability as well as circumvent the hepatic first-pass extractionof oral drug that is absorbed from the intestinal tract through theportal-hepatic venous system. This realization prompted the design of anIV Bu formulation to achieve controlled administration (Bhagwatwar etal., 1996; Andersson et al., 2000). The DMA-based IV Bu-formulation wasapproved by the US FDA in 1999. It has rapidly replaced oral Bu inpre-HSCT chemotherapy, mostly in the IV BuCy2 regimen (Andersson et al.,2002).

So far, IV BuCy2 has been compared with oral BuCy2 in 6 retrospectivestudies, all showing superiority of IV BuCy2 with regards to thedevelopment of VOD and early transplant-related mortality (Kashyap etal., 2002; Thall et al., 2004; Kim et al., 2005; Lee et al., 2005;Aggarwal et al., 2006; Dean et al., 2010). The introduction of IV Buwith Cy appeared to improve the safety of the Bu-Cy(2) regimen(s),however early regimen-related toxicity was/is still of concern. As notedabove, it had become apparent through the work of McDonald and coworkersthat Cy, when used in high doses in the pretransplant setting,contributed to overall hepatotoxicity (McDonald et al., 1993; McDonaldet al., 2003; DeLeve et al., 2002). The activated cytotoxic metabolitesof Cy (especially o-carboxyethylphosphoramide mustard and acrolein)likely contribute to VOD in the Bu-Cy2 conditioning regimen through theneed for GSH in their metabolic detoxification. As an extension of theseobservations, the risk for VOD could conceivably be decreased bysubstituting Cy with an immunosuppressive agent from a different classof drugs without hepatotoxicity, such as Fludarabine (Flu), which doesnot utilize GSH in its metabolism, and which is virtually non-toxic tothe liver. Thus, Russell and colleagues reported on a myeloablativeconditioning regimen using IV Bu-Flu and antithymocyte globulin (ATG) ina convenient once-daily dosing schedule (Russell et al., 2002). Insubsequent, disease-specific studies performed at the M.D. AndersonCancer Center (MDACC), Flu and IV Bu were also given once daily (De Limaet al., 2004; Andersson et al., 2008). Ninety-six patients with AML/MDSwere treated in this study where ATG was added only for matchedunrelated donor- and one antigen mismatched sibling donor-transplantpatients (De Lima et al., 2004). Stomatitis was commonly seen, and VODand neurological side effects were still encountered in a fraction ofpatients. The majority of patients in the first study and 18% in thesecond study had transient elevations of ALT, while about 10%experienced a significant increase in bilirubin as additional signs ofstress on liver function within one to two weeks after transplant(Russell et al., 2002; De Lima et al., 2004). Three of the first 166(1.8%) patients treated in these two trials developed clinicallysignificant VOD, and one of them died (0.6%). Neurotoxicity wasuncommon; 4% of patients developed a “hand-foot” syndrome and twopatients had seizures (Russell et al., 2002; De Lima et al., 2004;Andersson et al., 2008). Interestingly, the pattern of liver toxicityappears somewhat different than what was previously experienced withoral Bu; commonly there is a “silent hyperbilirubinemia” in about athird of the patients, having its onset within about a week of IV Budelivery, and if clinical VOD occurs, it now commonly happens at a latertime than what was previously encountered. Thus, clinically diagnosedVOD now occurred in a fraction of patients as late as two months afterthe HSCT (Andersson, unpublished data).

The parenteral Bu-formulation was developed to achieve 100%bioavailability with complete dose assurance, and to simultaneouslyeliminate the hepatic first-pass effect which may contribute to the highrisk of mortal liver failure after oral high-dose Bu (Bhagwatwar et al.,1996; U.S. Pat. Nos. 5,430,057; 5,559,148).

The currently available IV Bu formulation has a composite solventvehicle based on N,N-Dimethylacetamide (DMA) and Polyethylene-glycol 400(PEG/PEG400) (“DMA-Bu”) (Bhagwatwar et al., 1996; U.S. Pat. Nos.5,430,057; 5,559,148). Although several clinical studies confirmed thatthis DMA-Bu formulation is better tolerated and yields improved clinicalresults of patients transplanted for various types of leukemia andlymphomas (Kashyap et al., 2002; Thall et al., 2004; Kim et al., 2005;Lee et al., 2005; Aggarwal et al., 2006; Dean et al., 2010; DeLeve etal., 2002; Russell et al., 2002; De Lima et al., 2004; Andersson et al.,2008; Chae et al., 2007; Bredeson et al., 2008; Shimoni et al., 2006;Shimoni et al., 2010; Santarone et al., 2011), there was already fromthe early human trials apprehension about administering a large amountof DMA in humans, since DMA is recognized as a potentially quite toxicsolvent (Dwivedi, 2002; VICH Steering Committee, 2010; The Food and DrugAdministration, 2010; The Office of Environmental Health HazardAssessment, 2010). These concerns are justifiably augmented by thepossible additive or even synergistic (adverse) interaction(s) betweenDMA and Bu, since both agents exert significant metabolic stress on theliver. Even though the overall incidence of serious hepatic toxicity isdecreased when comparing the oral and IV Bu-Cy2 regimens (Kashyap etal., 2002; Thall et al., 2004; Kim et al., 2005; Lee et al., 2005;Aggarwal et al., 2006; Dean et al., 2010), there is a (sub-) group ofpatients who suffer serious, life-threatening, or even lethal, hepatictoxicity after receiving the IV Bu-Cy2 and IV Bu-Flu variant regimens(Kashyap et al., 2002; Thall et al., 2004; Kim et al., 2005; Lee et al.,2005; Aggarwal et al., 2006; Dean et al., 2010; Russell et al., 2002; DeLima et al., 2004; Andersson et al., 2008; Chae et al., 2007; Bredesonet al., 2008; Shimoni et al., 2006; Shimoni et al., 2010; Santarone etal., 2011).

It may be important to remember, that the hepatic toxicity profile of IVDMA-Bu is qualitatively somewhat different from that experienced withoral Bu; oral Bu toxicity is manifested as an early, progressiveincrease in bilirubin, ALT, and AST, typically emerging within the first10 days following oral Bu administration. This either rapidly progressesto life-threatening or lethal hepato-renal failure or, alternatively thepatient starts improving and is clinically significantly better at about3 weeks after transplantation; the likelihood for complete recovery isnow excellent (McDonald et al., 1993; Bearman, 1995; McDonald et al.,2003; DeLeve et al., 2002). In contrast, when the IV DMA-Bu is utilized,there is typically a high (about 30-40%) incidence of “silenthyperbilirubinemia” that appears within 10-14 days following IV DMA-Buadministration. This is likely to resolve in the next several days (upto about a week to ten days), but serious treatment-related hepatictoxicity, VOD, may instead manifest itself as late as 8-10 weekspost-HSCT (Russell et al., 2002; De Lima et al., 2004; Andersson et al.,2008). The inventors hypothesized that the changing clinical toxicitypattern may result from an adverse interaction between Bu and DMA. Thelatter solvent has demonstrated hepatic, renal and neurologic toxicityin humans, in addition to causing growth retardation and decreasedweight gain using rodents and logomorphs in experimental settings(Malley et al., 1995; Kennedy, 1986; Klimisch and Hellwig, 2000; Okudaet al., 2006; Valentine et al., 1997; Kennedy, 2001). In addition, thereis at least one report of serious toxic hepatitis with an incidence ofabout 3-5% in factory workers that were occupationally exposed to highconcentrations of vaporized DMA in a commercial plastics productionfacility (Choi et al., 2001). Finally, in a clinical study of DMA as ananti-cancer agent, the acute, dose-limiting toxicity of DMA was mentalconfusion/coma, and DMA has also been described as a hallucinogenicagent (Weiss et al., 1962a; Weiss et al., 1962b). The concern about (a)serious adverse interaction(s) between Bu and DMA led one group toinvestigate the possible clinical adverse interaction(s) when IV DMA-Buformulation is combined with Cy in pretransplant conditioning therapy(Hempel et al., 2007). These investigators concluded, that althoughthere might be justifiable concerns about (an) adverse interaction(s)between Bu and DMA and Cy, the available IV DMA-Bu formulation is stillsafer than oral Bu when used in pretransplant conditioning therapy(Hempel et al., 2007). Other investigators demonstrated that undercarefully controlled conditions Bu and DMA have a significant(synergistic) cytotoxic interaction (Sadeghi et al., 1999). It isconceivable, that a potentially serious adverse clinical interactionbetween the two agents is obscured by the naturally occurringinterindividual heterogeneity in drug metabolism in the clinicalsituation. Further, the only comparison that is possible when trying toidentify a suitable reference population for evaluation of clinicalsafety of IV DMA-Bu is the historical comparison with patients treatedwith oral Bu-based high-dose chemotherapy. Because of the excessivelyhigh risk for serious treatment-related complications after high-doseoral Bu, such a comparison will undoubtedly favor DMA-Bu, but it doesnot address the contribution of DMA to the overall toxicity profile ofIV DMA-Bu. Presently such an evaluation is not possible to perform,since the only available IV Bu formulation has a large amount of DMA inthe solvent vehicle.

When all available data are considered, it is apparent that inclusion of(a) solvent(s) that impose(s) metabolic stress on the liver, such asDMA, will likely increase the risk for clinically significant liver-and/or multiorgan toxicity, thereby increasing the overall risk to thepatient for treatment-regimen related morbidity and mortality. This riskis, however, downplayed by the use of a historical comparator group thatwas subjected to a significantly worse therapeutic alternative.

The well documented toxicity profile of DMA has rendered it adesignation as a Class II agent from the International Cooperation onHarmonization of Technical requirements for Registration of VeterinaryMedicinal Products. This designation means that DMA is an agent whoseutilization in manufacturing of pharmaceutical formulations should bestrictly limited and, if at all possible, it should be avoided (Dwivedi,2002; VICH Steering Committee, 2010; The Food and Drug Administration,2010; The Office of Environmental Health Hazard Assessment, 2010).

Therefore, based on the 1) mostly occupational literature reports ofserious DMA-induced normal-organ (liver) toxicity, and 2) the acutechanges in level of consciousness and/or hallucinations whenadministered in humans, 3) the occasional later occurring cases ofserious, life-threatening as well as lethal, liver toxicity experiencedwith use of the IV DMA-Bu formulation, and finally 4) the existingFDA-guidelines, there is a need to design an alternative parenteral Buformulation that is free from DMA. The availability of such aformulation would serve to further improve the clinical safety profileof parenterally administered Bu, such that its full therapeuticpotential can be experienced without added concern for serious normalorgan toxicity that is imposed by (a) component(s) of the compositesolvent vehicle.

SUMMARY OF THE INVENTION

Certain aspects of the present invention provide pharmaceutically stableand parenterally acceptable novel formulations of lipophilicpharmaceutical agents. Without wishing to be bound by theory, theformulations of the invention may be partly based on the principle ofcosolvency. Particularly the invention is based, at least in part, onthe discovery that a lipophilic pharmaceutical agent could be stable andsolubilized at a higher concentration in a non-aqueous solvent by aspecific cosolvency approach. The approach may involve the use of avolatile organic solvent to facilitate solubilization of the lipophilicagent in a non-aqueous solvent such as PEG400, followed by the removalof the volatile organic solvent to provide a non-aqueous composition ofthe lipophilic agent with improved solubility and stability. Optionally,such non-aqueous composition may be further diluted with an aqueoussolvent while the lipophilic agent could remain stable and solubilized.Examples of the compositions may be pharmaceutically acceptable,nontoxic, and stable for many hours at room temperature, such as thebusulfan formulation.

The invention relates to pharmaceutical formulations, and morespecifically, to parenteral formulations of lipophilic agents such asbusulfan (Bu), an azole agent like Posaconazole, Itraconazole or relatedanti-infectious agents. In certain aspects of the invention, parenteralformulations may be useful for treatment of any conditions or diseasesthat are sensitive or responsive to the lipophilic agents, including,but not limited to, the treatment and/or suppression of malignant orautoimmune diseases, for use in conditioning therapy of patientsundergoing hematopoietic stem cell transplantation (HSCT) or for thetreatment and/or suppression of systemic infections with yeast, moldsand other organisms that are sensitive to anti-infectious agents.

Such parenteral formulations could avoid the undesirable, erraticbioavailability, and unpredictable hepatic first pass extraction of oralpreparations and in view of being truly solubilized the agents are nowfree from the shortcomings experienced with the delivery of particulatematter, such as colloidal, nano-particular or micro-particularsuspensions, or microcrystalline suspensions of pharmaceutically activeagents. In a particular aspect, the busulfan formulation may abrogatethe concern for acute, as well as long-term, or chronic, toxicityrelated to the inclusion of the organic solvent N,N-dimethylacetamide(DMA), as being the major component in the only commercially availableparenteral Bu formulation.

Accordingly, one embodiment of the invention is directed to anon-aqueous, homogeneous solution comprising a solubilized lipophilicpharmaceutical agent and a non-aqueous amphiphilic solvent, such as anamphiphilic liquid polymeric solvent. Without wishing to be bound bytheory, it is contemplated that the agent may bind to the amphiphilicsolvent by electrostatic interactions to achieve high aqueous solubilityand stability. Formulations of the present invention are essentiallyfree of non-polymeric organic solvents, water and non-solubilizedparticles, wherein the solubilized lipophilic pharmaceutical agent has aconcentration of at least about 0.5 mg/mL, and further wherein thesolution remains stable and essentially free of non-solubilizedparticles for at least 40 days and preferably at least 60 days. Studiesare described herein below demonstrating exemplary formulations thatmaintain such properties when stored for at least 40 and even up to 60days at room temperature when tested at 5 mg pharmaceutical agent/ml ofamphiphilic liquid polymeric solvent.

In certain aspects, the solution of any formulation described herein maybe essentially free of DMA or other polymeric or non-polymeric organicsolvents. In particular aspects, the formulation may be essentially freeof water or obviate the need of the use of water in formulationpreparation. Non-solubilized particles, such as colloidal particles,nano-particles or micro-particles, or microcrystalline particles, mayalso be essentially non-existent in the solution of any formulationdescribed herein. The solution composition may optionally furthercomprise an aqueous diluent such as an aqueous infusion fluid, which maybe used to facilitate the subsequent systemic administration to amammal, preferably a human or a (large) domestic animal. In a furtheraspect, an aqueous, homogeneous, pharmaceutically-acceptable parenteralformulation may be prepared by a process comprising obtaining a solutiondescribed above and diluting the solution with a desired aqueousdiluent.

In certain aspects, the invention may be directed to compositions andmethods for parenteral formulation preparation. The novel solventvehicles of the invention may be used to facilitate parenteraladministration of other hard-to solubilize, aka “water-insoluble”,drugs. Accordingly, another embodiment of the invention includes acomposition for parenteral use comprising: a water-insoluble/lipophilicpharmaceutically active agent, and a first solvent, the first solventcomprising an organic solvent such as acetone or chloroform togetherwith a second amphiphilic solvent, such as PEG. The pharmaceuticallyactive agent may be dissolved in the first solvent, and aftersolubilization it may be mixed with the second solvent. The firstorganic (volatile) solvent may be then removed (e.g., evaporated undervacuum) and the pharmaceutically active agent may remainelectrostatically attracted and bound to, and stably dissolved in, thesecond solvent/PEG. The clinical use-composition optionally furthercomprises a secondary diluent such as an aqueous infusion fluid, such asnormal saline or dextrose in water, either by itself or pre-mixed with asmall amount (10-25%, v/v) of amphiphilic polymer such as PEG. Due tothe electrostatic attraction between the second amphiphilic solvent(PEG) and the pharmaceutically active agent this drug-PEG complex can bediluted in the aqueous diluent without immediate precipitation of thepharmaceutically active agent.

In a particular aspect, the composition may comprise Bu and a firstvolatile organic solvent such as acetone. The Bu may be dissolved in thevolatile organic solvent such as acetone and then mixed with anamphiphilic solvent such as PEG400. Subsequently, and taking advantageof the low boiling point of the volatile organic solvent, the volatileorganic solvent may be removed, e.g., by evaporation under vacuum at RT.At the end of this phase, the Bu could be completely and stablydissolved in the amphiphilic solvent such as PEG400. Prior to clinicaladministration, the composition may be optionally diluted with asecondary diluent such as an aqueous infusion fluid, e.g., normal saline(NS) or 5-10% dextrose in water (D5W, D10W), as final diluent(s).

The solubilized lipophilic pharmaceutical agent in any solution,composition or formulation described herein may have a concentration ofat least or up to about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL (or mol/L) orany intermediate ranges or numbers. In particular aspects, thesolubilized lipophilic pharmaceutical agent may have a concentration ofabout 1 to 10 mg/mL or about 3 to 9 mg/mL.

For example, the lipophilic pharmaceutical agents that can be usedherein include lipophilic compounds having solubility in an aqueoussolvent of less than about 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10 mg/mL (ormol/L) or any range derivable therein, preferably less than 10 mg/mL,more preferably less than about 1 mg/ml and even less than about 0.1mg/mL.

In certain aspects, formulations described herein could retain at least50, 60, 70, 80, 90, 95, 99, 99.9, 100% activity (or any value or rangederivable therein) of the pharmaceutical agents during or afterpreparation. For example, the novel Bu formulation retains full in vitrocytotoxic activity in tissue cultures utilizing continuously growinghuman leukemia cell lines as targets, demonstrating that the novel Buformulations do not lose cytotoxic activity due to chemical degradationor physical precipitation when solubilized. Formulations describedherein may be used intravascularly, and have been successfully used forintravenous (IV) administration in a murine model. Preliminarypharmacokinetics obtained in a mouse model with an exemplary formulationof the invention has yielded detectable cytotoxic Bu concentrations forseveral hours after administration.

Suitable lipophilic agents may be any poorly water-soluble, biologicallyactive agents or a salt, isomer, ester, ether or other derivativethereof, which include, but are not limited to, anticancer agents,antifungal agents, psychiatric agents such as analgesics, consciousnesslevel-altering agents such as anesthetic agents or hypnotics,nonsteroidal antiinflammatory agents, anthelminthics, antiacne agents,antianginal agents, antiarrhythmic agents, anti-asthma agents,antibacterial agents, anti-benign prostate hypertrophy agents,anticoagulants, antidepressants, antidiabetics, antiemetics,antiepileptics, antigout agents, antihypertensive agents,antiinflammatory agents, antimalarials, antimigraine agents,antimuscarinic agents, antineoplastic agents, antiobesity agents,antiosteoporosis agents, antiparkinsonian agents, antiproliferativeagents, antiprotozoal agents, antithyroid agents, antitussive agent,anti-urinary incontinence agents, antiviral agents, anxiolytic agents,appetite suppressants, beta-blockers, cardiac inotropic agents,chemotherapeutic drugs, cognition enhancers, contraceptives,corticosteroids, Cox-2 inhibitors, diuretics, erectile dysfunctionimprovement agents, expectorants, gastrointestinal agents, histaminereceptor antagonists, immunosuppressants, keratolytics, lipid regulatingagents, leukotriene inhibitors, macrolides, muscle relaxants,neuroleptics, nutritional agents, opioid analgesics, proteaseinhibitors, or sedatives.

Non-limiting examples of lipophilic agents may include7-Methoxypteridine, 7-Methylpteridine, abacavir, abafungin, abarelix,acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide,acetohexamide, acetretin, acrivastine, adenine, adenosine,alatrofloxacin, albendazole, albuterol, alclofenac, aldesleukin,alemtuzumab, alfuzosin, alitretinoin, allobarbital, allopurinol,all-transretinoic acid (ATRA), aloxiprin, alprazolam, alprenolol,altretamine, amifostine, amiloride, aminoglutethimide, aminopyrine,amiodarone HCl, amitriptyline, amlodipine, amobarbital, amodiaquine,amoxapine, amphetamine, amphotericin, amphotericin B, ampicillin,amprenavir, amsacrine, amylnitrate, amylobarbitone, anastrozole,anrinone, anthracene, anthracyclines, aprobarbital, arsenic trioxide,asparaginase, aspirin, astemizole, atenolol, atorvastatin, atovaquone,atrazine, atropine, atropine azathioprine, auranofin, azacitidine,azapropazone, azathioprine, azintamide, azithromycin, aztreonum,baclofen, barbitone, BCG live, beclamide, beclomethasone,bendroflumethiazide, benezepril, benidipine, benorylate, benperidol,bentazepam, benzamide, benzanthracene, benzathine penicillin, benzhexolHCl, benznidazole, benzodiazepines, benzoic acid, bepheniumhydroxynaphthoate, betamethasone, bevacizumab (avastin), bexarotene,bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene,bleomycin, bleomycin, bortezomib, brinzolamide, bromazepam,bromocriptine mesylate, bromperidol, brotizolam, budesonide, bumetanide,bupropion, busulfan, butalbital, butamben, butenafine HCl,butobarbitone, butobarbitone (butethal), butoconazole, butoconazolenitrate, butylparaben, caffeine, calcifediol, calciprotriene,calcitriol, calusterone, cambendazole, camphor, camptothecin,camptothecin analogs, candesartan, capecitabine, capsaicin, captopril,carbamazepine, carbimazole, carbofuran, carboplatin, carbromal,carimazole, carmustine, cefamandole, cefazolin, cefixime, ceftazidime,cefuroxime axetil, celecoxib, cephradine, cerivastatin, cetrizine,cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide,chlormethiazole, chloroquine, chlorothiazide, chlorpheniramine,chlorproguanil HCl, chlorpromazine, chlorpropamide, chlorprothixene,chlorpyrifos, chlortetracycline, chlorthalidone, chlorzoxazone,cholecalciferol, chrysene, cilostazol, cimetidine, cinnarizine,cinoxacin, ciprofibrate, ciprofloxacin HCl, cisapride, cisplatin,citalopram, cladribine, clarithromycin, clemastine fumarate, clioquinol,clobazam, clofarabine, clofazimine, clofibrate, clomiphene citrate,clomipramine, clonazepam, clopidogrel, clotiazepam, clotrimazole,clotrimazole, cloxacillin, clozapine, cocaine, codeine, colchicine,colistin, conjugated estrogens, corticosterone, cortisone, cortisoneacetate, cyclizine, cyclobarbital, cyclobenzaprine,cyclobutane-spirobarbiturate, cycloethane-spirobarbiturate,cycloheptane-spirobarbiturate, cyclohexane-spirobarbiturate,cyclopentane-spirobarbiturate, cyclophosphamide,cyclopropane-spirobarbiturate, cycloserine, cyclosporin, cyproheptadine,cyproheptadine HCl, cytarabine, cytosine, dacarbazine, dactinomycin,danazol, danthron, dantrolene sodium, dapsone, darbepoetin alfa,darodipine, daunorubicin, decoquinate, dehydroepiandrosterone,delavirdine, demeclocycline, denileukin, deoxycorticosterone,desoxymethasone, dexamethasone, dexamphetamine, dexchlorpheniramine,dexfenfluramine, dexrazoxane, dextropropoxyphene, diamorphine,diatrizoicacid, diazepam, diazoxide, dichlorophen, dichlorprop,diclofenac, dicumarol, didanosine, diflunisal, digitoxin, digoxin,dihydrocodeine, dihydroequilin, dihydroergotamine mesylate,diiodohydroxyquinoline, diltiazem HCl, diloxamide furoate,dimenhydrinate, dimorpholamine, dinitolmide, diosgenin, diphenoxylateHCl, diphenyl, dipyridamole, dirithromycin, disopyramide, disulfiram,diuron, docetaxel, domperidone, donepezil, doxazosin, doxazosin HCl,doxorubicin (neutral), doxorubicin HCl, doxycycline, dromostanolonepropionate, droperidol, dyphylline, echinocandins, econazole, econazolenitrate, efavirenz, ellipticine, enalapril, enlimomab, enoximone,epinephrine, epipodophyllotoxin derivatives, epirubicin, epoetinalfa,eposartan, equilenin, equilin, ergocalciferol, ergotamine tartrate,erlotinib, erythromycin, estradiol, estramustine, estriol, estrone,ethacrynic acid, ethambutol, ethinamate, ethionamide, ethopropazine HCl,ethyl-4-aminobenzoate (benzocaine), ethylparaben, ethinylestradiol,etodolac, etomidate, etoposide, etretinate, exemestane, felbamate,felodipine, fenbendazole, fenbuconazole, fenbufen, fenchlorphos,fenclofenac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium,fenoxycarb, fenpiclonil, fentanyl, fenticonazole, fexofenadine,filgrastim, finasteride, flecamide acetate, floxuridine, fludarabine,fluconazole, fluconazole, flucytosine, fludioxonil, fludrocortisone,fludrocortisone acetate, flufenamic acid, flunanisone, flunarizine HCl,flunisolide, flunitrazepam, fluocortolone, fluometuron, fluorene,fluorouracil, fluoxetine HCl, fluoxymesterone, flupenthixol decanoate,fluphenthixol decanoate, flurazepam, flurbiprofen, fluticasonepropionate, fluvastatin, folic acid, fosenopril, fosphenytoin sodium,frovatriptan, furosemide, fulvestrant, furazolidone, gabapentin, G-BHC(Lindane), gefitinib, gemcitabine, gemfibrozil, gemtuzumab, glafenine,glibenclamide, gliclazide, glimepiride, glipizide, glutethimide,glyburide, Glyceryltrinitrate (nitroglycerin), goserelin acetate,grepafloxacin, griseofulvin, guaifenesin, guanabenz acetate, guanine,halofantrine HCl, haloperidol, hydrochlorothiazide, heptabarbital,heroin, hesperetin, hexachlorobenzene, hexethal, histrelin acetate,hydrocortisone, hydroflumethiazide, hydroxyurea, hyoscyamine,hypoxanthine, ibritumomab, ibuprofen, idarubicin, idobutal, ifosfamide,ihydroequilenin, imatinib mesylate, imipenem, indapamide, indinavir,indomethacin, indoprofen, interferon alfa-2a, interferon alfa-2b,iodamide, iopanoic acid, iprodione, irbesartan, irinotecan,isavuconazole, isocarboxazid, isoconazole, isoguanine, isoniazid,isopropylbarbiturate, isoproturon, isosorbide dinitrate, isosorbidemononitrate, isradipine, itraconazole, itraconazole, itraconazole(Itra), ivermectin, ketoconazole, ketoprofen, ketorolac, khellin,labetalol, lamivudine, lamotrigine, lanatoside C, lanosprazole, L-DOPA,leflunomide, lenalidomide, letrozole, leucovorin, leuprolide acetate,levamisole, levofloxacin, lidocaine, linuron, lisinopril, lomefloxacin,lomustine, loperamide, loratadine, lorazepam, lorefloxacin,lormetazepam, losartan mesylate, lovastatin, lysuride maleate,Maprotiline HCl, mazindol, mebendazole, Meclizine HCl, meclofenamicacid, medazepam, medigoxin, medroxyprogesterone acetate, mefenamic acid,Mefloquine HCl, megestrol acetate, melphalan, mepenzolate bromide,meprobamate, meptazinol, mercaptopurine, mesalazine, mesna,mesoridazine, mestranol, methadone, methaqualone, methocarbamol,methoin, methotrexate, methoxsalen, methsuximide, methyclothiazide,methylphenidate, methylphenobarbitone, methyl-p-hydroxybenzoate,methylprednisolone, methyltestosterone, methyprylon, methysergidemaleate, metoclopramide, metolazone, metoprolol, metronidazole,Mianserin HCl, miconazole, midazolam, mifepristone, miglitol,minocycline, minoxidil, mitomycin C, mitotane, mitoxantrone,mofetilmycophenolate, molindone, montelukast, morphine, MoxifloxacinHCl, nabumetone, nadolol, nalbuphine, nalidixic acid, nandrolone,naphthacene, naphthalene, naproxen, naratriptan HCl, natamycin,nelarabine, nelfinavir, nevirapine, nicardipine HCl, nicotin amide,nicotinic acid, nicoumalone, nifedipine, nilutamide, nimodipine,nimorazole, nisoldipine, nitrazepam, nitrofurantoin, nitrofurazone,nizatidine, nofetumomab, norethisterone, norfloxacin, norgestrel,nortriptyline HCl, nystatin, oestradiol, ofloxacin, olanzapine,omeprazole, omoconazole, ondansetron HCl, oprelvekin, ornidazole,oxaliplatin, oxamniquine, oxantelembonate, oxaprozin, oxatomide,oxazepam, oxcarbazepine, oxfendazole, oxiconazole, oxprenolol,oxyphenbutazone, oxyphencyclimine HCl, paclitaxel, palifermin,pamidronate, p-aminosalicylic acid, pantoprazole, paramethadione,paroxetine HCl, pegademase, pegaspargase, pegfilgrastim,pemetrexeddisodium, penicillamine, pentaerythritol tetranitrate,pentazocin, pentazocine, pentobarbital, pentobarbitone, pentostatin,pentoxifylline, perphenazine, perphenazine pimozide, perylene,phenacemide, phenacetin, phenanthrene, phenindione, phenobarbital,phenolbarbitone, phenolphthalein, phenoxybenzamine, phenoxybenzamineHCl, phenoxymethyl penicillin, phensuximide, phenylbutazone, phenytoin,pindolol, pioglitazone, pipobroman, piroxicam, pizotifen maleate,platinum compounds, plicamycin, polyenes, polymyxin B, porfimersodium,posaconazole (Posa), pramipexole, prasterone, pravastatin, praziquantel,prazosin, prazosin HCl, prednisolone, prednisone, primidone,probarbital, probenecid, probucol, procarbazine, prochlorperazine,progesterone, proguanil HCl, promethazine, propofol, propoxur,propranolol, propylparaben, propylthiouracil, prostaglandin,pseudoephedrine, pteridine-2-methyl-thiol, pteridine-2-thiol,pteridine-4-methyl-thiol, pteridine-4-thiol, pteridine-7-methyl-thiol,pteridine-7-thiol, pyrantelembonate, pyrazinamide, pyrene,pyridostigmine, pyrimethamine, quetiapine, quinacrine, quinapril,quinidine, quinidine sulfate, quinine, quininesulfate, rabeprazolesodium, ranitidine HCl, rasburicase, ravuconazole, repaglinide, reposal,reserpine, retinoids, rifabutine, rifampicin, rifapentine, rimexolone,risperidone, ritonavir, rituximab, rizatriptan benzoate, rofecoxib,ropinirole HCl, rosiglitazone, saccharin, salbutamol, salicylamide,salicylic acid, saquinavir, sargramostim, secbutabarbital, secobarbital,sertaconazole, sertindole, sertraline HCl, simvastatin, sirolimus,sorafenib, sparfloxacin, spiramycin, spironolactone, stanolone,stanozolol, stavudine, stilbestrol, streptozocin, strychnine,sulconazole, sulconazole nitrate, sulfacetamide, sulfadiazine,sulfamerazine, sulfamethazine, sulfamethoxazole, sulfanilamide,sulfathiazole, sulindac, sulphabenzamide, sulphacetamide, sulphadiazine,sulphadoxine, sulphafurazole, sulphamerazine, sulpha-methoxazole,sulphapyridine, sulphasalazine, sulphinpyrazone, sulpiride, sulthiame,sumatriptan succinate, sunitinib maleate, tacrine, tacrolimus, talbutal,tamoxifen citrate, tamulosin, targretin, taxanes, tazarotene,telmisartan, temazepam, temozolomide, teniposide, tenoxicam, terazosin,terazosin HCl, terbinafine HCl, terbutaline sulfate, terconazole,terfenadine, testolactone, testosterone, tetracycline,tetrahydrocannabinol, tetroxoprim, thalidomide, thebaine, theobromine,theophylline, thiabendazole, thiamphenicol, thioguanine, thioridazine,thiotepa, thotoin, thymine, tiagabine HCl, tibolone, ticlopidine,tinidazole, tioconazole, tirofiban, tizanidine HCl, tolazamide,tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab,tramadol, trastuzumab, trazodone HCl, tretinoin, triamcinolone,triamterene, triazolam, triazoles, triflupromazine, trimethoprim,trimipramine maleate, triphenylene, troglitazone, tromethamine,tropicamide, trovafloxacin, tybamate, ubidecarenone (coenzyme Q10),undecenoic acid, uracil, uracil mustard, uric acid, valproic acid,valrubicin, valsartan, vancomycin, venlafaxine HCl, vigabatrin,vinbarbital, vinblastine, vincristine, vinorelbine, voriconazole,xanthine, zafirlukast, zidovudine, zileuton, zoledronate, zoledronicacid, zolmitriptan, zolpidem, and zopiclone.

In particular aspects, the agents may be busulfan, taxane or otheranticancer agents; or alternatively, itraconazole (Itra) andposaconazole (Posa) or other members of the general class of azolecompounds. Exemplary antifungal azoles include a) imidazoles such asmiconazole, ketoconazole, clotrimazole, econazole, omoconazole,bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole,sertaconazole, sulconazole and tioconazole, b) triazoles such asfluconazole, itraconazole, isavuconazole, ravuconazole, Posaconazole,voriconazole, terconazole and c) thiazoles such as abafungin. Otherdrugs that can be solubilized with this approach include, but are notlimited to, hyperthyroid drugs such as carimazole, anticancer agentslike cytotoxic agents such as epipodophyllotoxin derivatives, taxanes,bleomycin, anthracyclines, as well as platinum compounds andcamptothecin analogs. They may also include other antifungalantibiotics, such as poorly water-soluble echinocandins, polyenes (e.g.,Amphotericin B and Natamycin) as well as antibacterial agents (e.g.,polymyxin B and colistin), and anti-viral drugs. The agents may alsoinclude a psychiatric agent such as an antipsychotic, anti-depressiveagent, or analgesic and/or tranquilizing agents such as benzodiazepines.The agents may also include a consciousness level-altering agent or ananesthetic agent, such as propofol. In a broader aspect, the presentinvention may provide methods to safely solubilize and administer manypoorly water-soluble, pharmacologically active agents.

As an additional advantage, any compositions described herein mayobviate the need of a surfactant, thus a polyethylene glycol (PEG) fattyacid ester surfactant (but not PEG itself) or other surfactants may notbe used in certain aspects. In other aspects, a surfactant known in theart may be used.

An amphiphilic liquid polymeric solvent may be used to provide/simulatea non-polar/lipophilic milieu. The amphiphilic liquid polymeric solventmay be of a single polymer type, or have at least two polymer types insome aspects. For example, the amphiphilic liquid polymeric solvent maybe a PEG solvent such as PEG-100, -200, -300, -400, -800, -1000, and thelike. A particular example may be PEG-400. The PEG used herein mayexclude any PEG that is in a solid state at a selected temperature suchas room temperature, body temperature or a temperature of at least,about or at most 5, 10, 15, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80° C., or any rangeor value derivable therein, such as PEG with a high molecular weight(average molecular weight of at least or more than 1600, 2000, 3000,4000, 5000, 6000, 10,000 dalton or any intermediate ranges). Forexample, the liquid solvent may be PEG-800 or PEG-1000 as they areliquid at body temperature.

To facilitate solubilization of the lipophilic agents, the compositioninvolving the lipophilic agents may further comprise a protonating agentto facilitate protonation of the reactive groups in lipophilic agents.For example, the protonating agent is an acid, alcohol or acidifiedalcohol (such as benzyl alcohol, and/or acidified ethanol). Non-limitingexamples of acid include HCl, citric acid, acetic acid or glutamic acidor other inorganic acids or organic acids known in the art. Thecomposition may have an acidic pH, such as a pH value or range derivedfrom a pH of from about 0.5, 1, 2, 3, 4, 5, 6, 6.5, and 6.9, preferablyin a range from about 1 to about 6.

The invention also includes a method of preparing a non-aqueous,homogeneous solution described above, comprising the steps of: obtaininga first non-aqueous, homogeneous solution comprising a lipophilicpharmaceutical agent, an amphiphilic liquid polymeric solvent and avolatile organic solvent, and removing the volatile organic solvent fromthe first solution to form a second non-aqueous, homogeneous solution asdescribed herein (“stock solution” or may be used in final clinicaluse). The volatile organic solvent may be used to facilitate binding ofthe lipophilic agents to the polymeric solvent via electrostaticinteractions. Non-limiting examples may include acetone, chloroform,aliphatic hydrocarbons, ethyl acetate, glycol ethers, diethyl-ether, orethanol. A particular example may be acetone. The method may be definedas a method for preparing an aqueous, homogeneous,pharmaceutically-acceptable parenteral formulation as it may furthercomprise diluting the second solution described above with a desiredaqueous diluent to produce a final clinical use-formulation.

In further aspects, the volume or weight ratio of the volatile organicsolvent to the amphiphilic liquid polymeric solvent may be from about100:1 to 1:100, or particularly, 1:1, 1:2, 1:3, 1:5, 1:10, 1:20, 1:30,1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or any range derivable therein. Tofacilitate the interaction between the reactive groups in the lipophilicagents and amphiphilic solvents, the volatile organic solvent or thedesired aqueous diluent may be acidified. The method may furthercomprise storing any of the compositions for at least 1, 2, 3, 4, 5, 6,7 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 weeks, 4, 5, 6, 7, 8, 9, 10,11, 12 months, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or any value or rangederivable therein.

Any of the method steps, such as removal of the volatile organicsolvents or storage of any compositions, may be performed at atemperature of at least, about or at most 5, 10, 15, 20, 25, 30, 40, 45,50, 55, 60, 65, 70, 75, 80° C., or any range or value derivable therein.In a particular aspect, the temperature may be room temperature. Theremoving method may include any method that is known to remove avolatile organic solvent, such as evaporation, more particularly,vacuum-assisted evaporation. The removal may be extended to extract theprotonating agent.

After removal of the volatile organic solvent, the composition may bestable for at least 1, 2, 3, 4, 5, 6, 7 days, 2, 3, 4, 5, 6, 7, 8, 9,10, 12 weeks, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 2, 3, 4, 5, 6, 7, 8,9, 10 years or any value or range derivable therein. The composition maybe further diluted with a desired aqueous diluent to facilitate itsclinical administration. For example, the aqueous diluent may be aninfusion fluid selected from the group consisting of normal saline,dextrose in water, and a lipid-based infusion emulsion fluid. Inparticular, the aqueous infusion fluid may be 0.9% sodium chloride (NS),or 5% or 10% dextrose in water (D5W and D10W, respectively), or anaqueous lipid emulsion such as Intralipid™, or Liposyn™. In a furtheraspect, such an aqueous diluent may be modified by the addition of aprotonating agent or with a small amount of PEG as described above. Suchmodification of the diluent may be preferred if the protonating agent isremoved from the stock solution. The resulting stable, final useformulation may contain the dissolved pharmaceutical agent that,dissolved at room temperature (RT), remains stable for an extended timeto allow convenient handling and administration to the patients.

In further aspects, the invention includes a method of preparing awater-insoluble/lipophilic pharmaceutically active agent for parenteraluse comprising the steps of: solubilizing the pharmaceutically activeagent in a(n) (volatile) organic solvent, mixing it with a second,non-volatile hydrophobic agent. The second solvent may preferably haveamphiphilic properties, such as PEG. The method may further compriseevaporating off the more volatile organic solvent component under vacuumsuch that a local electrostatic attraction arises that binds thepharmaceutically active agent to the secondary amphiphilic solvent.Physical precipitation of the pharmaceutically active agent may therebybe prevented, thus producing a stock formulation. In a further aspect,the method may comprise mixing the dissolved pharmaceutically activeagent/amphiphilic solvent (e.g., PEG) complex with a final aqueousdiluent to provide a clinical use-formulation that can be administeredparenterally. For example, the organic solvent is acetone or chloroform,or diethylether, with or without addition of a small amount of an acidto facilitate protonation of the pharmaceutically active agent toincrease the electrostatic attraction to the secondary solvent.Preferably the secondary amphiphilic solvent is a polymer such as PEG.The pharmaceutically active agent may be a bifunctional DNA-alkylatingagent such as busulfan (Bu) or, alternatively, it can be anantimicrobial agent such as an azole compound used to treat fungal orparasitic infections, or a hypnotic or sedative agent used inpsychiatric or anesthetic settings, or alternatively it can be an agentused for symptom control such as an anesthetic or a consciousness-levelaltering agent such as a general anesthetic. Further, to increase thestable electrostatic attraction between the pharmaceutically activeagent and the amphiphilic solvent such as PEG, the vacuum-extraction maybe significantly extended to remove excess (free) acid from thedrug/PEG-complex. Finally, the method may comprise the step of mixingthe stock formulation with a secondary diluent, such as an aqueousinfusion fluid, to allow the administration of the pharmaceuticallyactive agent in a domestic animal or more preferably, in a human.

The invention may also include a method for treating a subject having adisease or condition sensitive or responsive to an lipophilicpharmaceutical agent, comprising: parenterally administering to thesubject a therapeutically effective dissolved amount of a compositioncomprising a solution or a formulation described above, wherein solutionor formulation has the lipophilic pharmaceutical agent to which that thedisease or condition is sensitive or responsive.

In a particular aspect, the invention also includes a method fortreating a disease sensitive or responsive to Bu comprising:parenterally administering a therapeutically effective amount of a Bucomposition to the patient. The Bu composition may be prepared bydissolving Bu in a first solvent comprising a volatile organic solvent,preferably acetone, then mixing the solution with a second amphiphilicsolvent, preferably PEG, subsequently evaporating the first organicsolvent under vacuum to create a stock-formulation of Bu in PEG, andoptionally diluting with a secondary aqueous diluent, such as an aqueousinfusion fluid.

Still another embodiment of the invention is directed to a method forparenterally administering Bu to a patient comprising: providing Bu inan organic, volatile hydrophobic solvent, subsequently mixed with asecond amphiphilic, non-volatile solvent; evaporating the firsthydrophobic solvent to produce a Bu stock formulation that can be eitherdirectly administered to the patient, or mixing the stock formulationwith a secondary aqueous diluent to form an infusion fluid; andadministering the infusion fluid to a patient. For example, the first,volatile organic solvent is acetone, and the secondary amphiphilicsolvent is PEG400.

The routes of administration may include, but are not limited to,administration intravascularly, intracavitarily, intrathecally,subcutaneously, intramuscularly, or topically. The subject may be amammal, particularly a domestic animal or a human.

In certain aspects, the subject has a cancer or a need for conditioningthe subject to perform a bone marrow transplantation or a hematopoieticprogenitor cell transplantation and the lipophilic pharmaceutical agentis busulfan. In other aspects, the subject has a fungal, yeast or molddisease and the lipophilic pharmaceutical agent is an azole agent. Infurther aspects, the subject has a psychiatric ailment or a need forsymptomatic control and the lipophilic pharmaceutical agent is apsychiatric agent, such as an antipsychotic, anti-depressive agent, oran analgesic agent. The subject has a need to alter the level ofconsciousness or to induce general anesthesia or conscious sedation andthe lipophilic pharmaceutical agent is a consciousness level-altering oran anesthetic agent such as propofol.

Other objects and advantages of the invention are set forth in part inthe description which follows and, in part, will be obvious from thisdescription, or may be learned from the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein.

FIGS. 1A-B. (A) A graph showing the stability of busulfan at roomtemperature in the final use-formulation of Bu/VE-acetone/PEG (i.e.,prototype stock solvent vehicle) containing Bu at an approximateconcentration of 5 mg/mL after vacuum extraction of acetone. (B) Thestock formulation is diluted with D5W to 1 mg/mL (top), and to 0.5 mg/mL(lower). The X-axis represents the time in hours, and the Y-axisrepresents the measured concentration in mg/mL.

FIG. 2. Standard curve of busulfan concentration vs. area under thecurve (AUC; area under the curve, term used to denote the actualmeasured area of a peak in a chromatogram, and also for the area underthe plasma concentration vs. time curve over several hours afteradministration of a drug to an animal or human being) for thehigh-pressure liquid chromatography (HPLC) assay used in the in vitrostability and in vivo pharmacology studies. The X-axis showsconcentration in μg/mL, and the Y-axis shows the AUC. An analogousstandard curve was prepared for the pharmacology studies.

FIG. 3. Chromatograms obtained from the HPLC assay in the stabilitystudies. The inventors used the Waters Nova-Pak C18 column, (4-μm beadsize; 150 mm×3.9 mm). The injected sample volume was 30 μL. The HPLCconditions are described in Example 1.

FIG. 4. A graph showing the hemolytic potential of the use-formulationof Bu/VE-acetone/PEG/D5W, and the same formulation (“solvent”) withoutbusulfan. The x-axis shows the solvent content in volume percent (v/v).The y-axis shows the calculated fraction of unhemolyzed red blood cells.

FIGS. 5A-C. Graph depicting the cytotoxic activity of busulfan in thePEG/D5W clinical use-formulation against the human cell lines KBM-3 (A)(Andersson et al., 1992) and KBM-7 (B) (Andersson et al., 1987;Andersson et al., 1995), assessed in vitro with the MTT assay. TheX-axis shows the Bu concentration in μg/mL; the Y-axis shows thecalculated cell survival fraction. As a positive control served cellsexposed in parallel to busulfan in DMSO. (C) shows the cytotoxicactivity of DMA alone in the MTT assay at the highest concentrationachieved when DMA-Bu was used as a positive control in the cell linesKBM3, KBM7, B5/Bu250-6, and in the OCI-AML3 (Wang et al., 1991). Thelatter findings correspond to a concentration which can be achieved whenDMA-Bu is used for pre-HSCT therapy with repeated dosing over 3-4 days.

FIG. 6. Sensitivity of three of the cell lines to Bu in DMSO and in thenew formulation relative to the cytotoxic effects reached with theDMA-Bu formulation. Of note is the significantly higher toxicity/lowersurvival fraction at increasing Bu concentrations with the DMA-Buformulation, and in particular in the KBM-3 cell line the contributionof DMA to overall cytotoxicity is significant. It appears from the datathat the effects of DMA and Bu are synergistic rather than additive(Chou and Talalay, 1984). In contrast, the current novel formulation andthe DMSO-Bu reference formulation exert virtually identical cytotoxiceffects in all tested cell lines, and there is no added toxic effect(s)from the solvent vehicle.

FIGS. 7A-C. Chromatograms of plasma samples extracted as described underExample 3 and then analyzed with HPLC. (A) The upper panel shows a blankplasma sample, (B) the middle panel shows a human plasma sample spikedwith busulfan in the new formulation (prototype use-solvent vehicle ofBu/VE-acetone/PEG/D5W) to 10 μg/mL, with a retention time ofapproximately 2.8 minutes. (C) The lower panel shows a chromatogram fromthe pharmacology study, where a mouse was injected with busulfan at 10mg/kg. The chromatogram was from a sample drawn 20 minutes after druginjection.

FIG. 8. Graph showing the change in plasma concentration over 4 hoursafter injection of 10 mg/kg of busulfan in mice. The X-axis shows thetime after dosing in hours. The Y-axis shows the concentration ofbusulfan in μg/mL plasma. The apparent busulfan half-life is in theapproximate range of 2.5-3.5 hours under the conditions used with thisnew formulation, similar to what has previously been reported for theDMA-Bu in rats and in humans (Bhagwatwar et al., 1996; Russell et al.,2002; De Lima et al., 2004 Madden et al., 2007).

FIGS. 9A-B. Stability of (A) Itra and (B) Posa in a variant formulationover a 3-week period at RT.

FIGS. 10A-B. Stability of (A) Itra and (B) Posa in the finaluse-formulations diluted in D5W.

FIG. 11. Photograph of in vitro sensitivity test of Aspergillus speciesto Itraconazole in the new formulation, for details see text.

FIGS. 12A-D. Chromatograms of Itra and Posa from the HPLC as plasmaalone, and plasma spiked with Itra and Posa in the stability studies.

FIGS. 13A-C. Chromatograms of blank plasma (upper panel), Posa afterspiking of human plasma (middle panel), as well as Posa in a sampleobtained 2 hours after IV injection of 5 mg/kg of Posa in mice (lowerpanel) as described under the experimental protocol in the text.

FIGS. 14A-B. Plasma concentrations after injection of Itra (FIG. 14A;over 2 hours) and Posa (FIG. 14B; over 30 hours) injected at a dose of 5mg/kg slowly IV (over 3-4 min) as described under the methods in thetext. The plasma concentrations are in a similar range as previouslydescribed in humans treated with the corresponding oral drugs in aclinical setting. The figure shows the average result of 2 differentexperiments, the individual time points and concentrations are detailedin the accompanying table.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Certain aspects of the present invention are directed to novelformulations containing lipophilic agents such as busulfan oranti-infectious agents, preferably belonging to the general class ofcompounds described as azoles, that may be administered parenterally. Anaspect of the invention provides for a solubilized lipophilic agent incomplex, pharmaceutically acceptable vehicles such that the dissolvedagent remains physically and chemically stable for prolonged time. Theinvention allows for parenteral administration of the drug in dosesnecessary to obtain significant pharmaceutical effects such as cytotoxicand immunosuppressive effects in subjects like humans and domesticanimals without undue toxicity from any component of the used solventvehicle. Exemplary embodiments of the invention allow for theparenteral, e.g. intravascular or intrathecal or intracavitaryadministration of solubilized agents to increase the safety of clinicaldrug administration. As a result, an improved control of diseases thatare sensitive to this agent such as malignant and autoimmune diseasesmay be achieved.

In certain aspects, there may be provided a method of preparing (a)hard-to-solubilize, “water-insoluble” or lipophilic pharmaceuticallyactive agent(s) for parenteral use. Suitable lipophilic pharmaceuticallyactive agents may include busulfan, azole agents such as Itra and Posa,or any lipophilic agents known in the art, as exemplified herein.Certain aspects of the present invention, which may be based on theprinciple of cosolvency but without wishing to be bound by theory, use anovel series of composite diluent vehicles to solubilize lipophilicagents, such as busulfan, itraconazole (Itra) and posaconazole (Posa),without affecting their pharmaceutical activity while improving aqueoussolubility and stability. Further, the preferred solvents are, in theproposed concentrations and total doses used, nontoxic and safe forhuman and mammalians, most preferably in humans and domestic animals.

The methods may first comprise dissolving the pharmaceutically activeagent in a primary volatile hydrophobic solvent followed by admixture ofa second non-volatile amphiphilic solvent. The methods may furthercomprise removing (e.g., by vacuum extraction) the primary volatilesolvent to provide a clinically acceptable stock formulation comprisingthe agent and the amphiphilic solvent. The methods may optionallycomprise diluting this stock formulation with an aqueous solvent, suchas an infusion fluid like D5W or D10W, or NS. Preferably, the primaryvolatile solvent is acetone and the second amphiphilic solvent isPEG-400.

In addition to acetone and PEG, other organic solvents may be used toform the solvent vehicle without departing from the spirit and scope ofthe invention. A volatile solvent can be a single solvent or a mixtureof solvents that are volatile, including water and solvents that aremore volatile than water. Non-limiting examples of volatile solventsthat can be used in the present invention include acetone, chloroform,aliphatic hydrocarbons, ethyl acetate, glycol ethers, diethyl-ether,iso-amyl acetate, denatured alcohol, methanol, ethanol, isopropylalcohol, propanol, C4-C6 hydrocarbons, butane, isobutene, pentane,hexane, acetone, chlorobutanol, ethyl acetate,fluro-chloro-hydrocarbons, turpentine, methyl ethyl ketone, methylether, hydrofluorocarbons, ethyl ether, 1,1,1,2 tetrafluorethane,1,1,1,2,3,3,3-heptafluoropropane, 1,1,1,3,3,3 hexafluoropropane, andcombinations thereof. The volatile solvent may be substantially removedby evaporation to form a homogenous solution comprising the agent andthe amphiphilic liquid solvent essentially free of the volatile solvent.The use of the term “substantially” when referring to the removal of thevolatile solvents means that a majority of the volatile solvent(s) whichwas/were included in the initial formulation has/have been removed.

Non-volatile amphiphilic solvents may be one or more solvents that areless volatile than water. Similarly, a non-volatile solvent is definedas a solvent that is less volatile than water. Preferably, thenon-volatile solvent may contain substances that are liquid at roomtemperatures. After evaporation of the volatile solvent, most of thenon-volatile solvent system should remain in a homogenous solutioncomprising the lipophilic agent.

For some hydrophobic agents, in particular drugs that contain unchargedreactive functional (amino) groups, the electrostatic attraction betweenthe amphiphilic solvent such as PEG and the pharmaceutically activeagent may be augmented by adding a protonating agent such as an organicacid or HCl, or an alcohol such as benzylalcohol, to the first organicsolvent prior to mixing the solubilized drug with PEG. After removal ofthe first organic solvent the final use-formulation is arrived to by theadmixture of a clinically acceptable aqueous infusion fluid. If thelatter preferred step of using a protonating agent (acid) to increasethe electrostatic attraction between the pharmaceutically active agentand PEG then a prolonged vacuum-extraction may be utilized to assureextraction not only of the first organic solvent but also of remainingfree acid. Said removal of excess free acid will allow for extending theshelf-life of the pharmaceutically active agent when bound to theamphiphilic agent such as PEG. If the latter approach is utilized thenit is preferred that the reconstitution prior to in vivo administrationbe done using acidified D5W or D10W as the final diluent to maintainoptimal electrostatic attraction between the pharmaceutically activeagent and PEG to prevent precipitation of the agent prior to parenteraladministration.

As shown in the Examples, Busulfan (Bu) and two prototype antifungalazole agents were successfully formulated for parenteral administration,utilizing a novel solvent system approach. For example, the lipophilicdrugs may be solubilized in a volatile primary solvent vehicle mixedwith (a) secondary non-volatile, non-toxic amphiphilic polymersolvent(s). In a particular embodiment, the primary solvent may be avolatile organic solvent such as acetone, or chloroform. Subsequentlythe first volatile solvent may be removed, for example, by vacuumextraction, but the drugs may remain solubilized in a solution of thepolymer solvent. An optional diluent, e.g., a clinically acceptableinfusion fluid such as D5W, may allow for dilution of thevacuum-extracted solution into clinical use-formulations that are stablefor many (more than 12) hours at RT. As an added benefit of the newformulation(s) should also be mentioned that when diluted in the finaluse-formulation, the current composition allows lipophilic drugs to beadministered with improved solubility and stability in the finaluse-formulation. For example, Bu could be administered at a higherconcentration of at least 1 mg/mL as compared with the currently usedDMA-Bu at only 0.5 mg/mL and it has a more extended stability at RT (atleast about 15 hours vs. 6 hours for DMA-Bu), both of which contributeto improved patient safety and convenience in routine pharmacy drughandling.

In a particular embodiment of the invention, Bu may be dissolved using avolatile solvent such as acetone, and then combined with an amphiphilicsolvent such as PEG as the composite vehicle or solvent system. If thissolubilized Bu/acetone/PEG mixture is mixed with water, the Bu remainsin solution without precipitation for several hours. However, due to thetoxicity of acetone to mammalian tissues (Dwivedi, 2002; VICH SteeringCommittee, 2010; The Food and Drug Administration, 2010; The Office ofEnvironmental Health Hazard Assessment, 2010), the acetone may bepreferably removed under vacuum, such that the pharmaceutically activeagent may become electrostatically attracted/bound to PEG in annon-aqueous solution, a procedure that has not been previouslydocumented. The novel exemplary Bu stock formulations (i.e. prior to theaddition of the secondary/final diluent) may be stable for many weeks atroom temperature, are simple to handle, and provide for reliable andeasily controlled, consistent dose administration. Prior toadministration such as parenteral administration, the non-aqueous stocksolution may be then (optionally) diluted in a secondary diluent such asa readily available infusion fluid, e.g., 5-10% dextrose-in-water (D5Wand D10W, respectively). The non-aqueous stock solution (Bu-PEG variantcompositions) may be miscible in secondary/final aqueous diluents, orroutinely available aqueous infusion fluids, e.g. 0.9% sodium chloride(normal saline, NS), and D5W, as well as a stock solution (such as D5W)with 10-25% (v/v) of an amphiphilic polymer (e.g., PEG). Such terminaldiluents/infusion fluids are typical examples of vehicles used tosolubilize pharmacologically active agents for human administration,alone or in combination with other drugs. The admixture of a smallamount of an amphiphilic polymer to the aqueous infusion fluid willfurther stabilize the lipophilic compound when prepared for parenteralinfusion.

Busulfan as an orally administered anticancer agent has previously beenextensively investigated in humans, and in the last decade these datahave been supplemented with results obtained with the DMA-basedparenteral formulation; Busulfan has well documented cytotoxic,myelosuppressive, as well as immunosuppressive properties in bothclinical and experimental settings. Unfortunately, Bu is a poorlywater-soluble DNA-alkylating agent with exceedingly low solubility inphysiologically acceptable aqueous solvents that would be compatiblewith human parenteral administration. Prior to the present invention,the only available administration forms have been an oral preparationand the DMA-based parenteral formulation. A parenteral formulation of Buthat is free from the risk of adverse events related to the highDMA-content has not been available. Such a parenteral Bu formulationwould be useful to evaluate Bu by itself and in combination with otherdrugs as part of individualized therapy for systemic malignant andautoimmune disorders as well as when profound long-termimmunosuppression is desirable, for instance as required in preparationfor (allogeneic) hematopoietic stem cell transplantation (HSCT) for bothmalignant and non-malignant, e.g. most commonly inborn/geneticdisorders. A parenteral formulation may need complete dose assurance andguaranteed 100% bioavailability.

As discussed in the Examples below, novel vehicles have been discoveredwhich achieve the stable, pharmaceutically acceptable solubilization ofBu, thereby making it safe to administer this drug intravascularlywithout the undue toxicity of DMA, something previously unattainable.The data in the Examples demonstrate that the novel Bu formulations maybe used for parenteral treatment of malignant and advanced autoimmunedisorders, as well as in conditioning therapy for HSCT.

Busulfan is very hydrophobic/lipophilic, and for practical purposeinsoluble in water and PEG. The use of a volatile hydrophobic solventsuch as acetone dissolves it and through the addition of an amphiphilicliquid solvent such as PEG with subsequent evacuation of the volatilesolvent the Bu may be contemplated to be electrostaticallystabilized/bound to the amphiphilic liquid solvent such as PEG, suchthat it tolerates further dilution in an aqueous diluent or blood plasmawithout imminent physical precipitation or chemical degradation. Thestability of the new formulation may permit combined handling andinfusion times in excess of 12 hours without significant loss of drugactivity.

As shown in the Examples, the described acetone-PEG-based vehicles weresuccessfully used to dissolve Bu at concentrations ranging from 0.1 toat least 10 mg/mL. This range is broad enough to cover theadministration of doses necessary to yield cytotoxic concentrations invivo when treating malignancies sensitive to this drug. Similarly, thisrange permits administration of the dose(s) necessary to achieveeffective immunosuppression in patients with autoimmune disorders andthose undergoing pre-HSCT conditioning therapy.

The data obtained in the Examples further demonstrate that stable Buformulation(s) may allow parenteral treatment of systemic malignant andautoimmune diseases. This preparation may consistently provide 100% drugbioavailability, and it may allow circumvention of the hepaticfirst-pass extraction. After a brief IV injection, the plasma Buconcentrations clearly reach, and for extended time remain in, thecytotoxic range as established by the in vitro studies of its cytotoxicactivity against human malignant cell lines, and these concentrationsalso compare favorably with several investigations that utilized eitheroral Bu or the DMA-Bu formulation (Slattery et al., 1997; Dix et al.,1996; Hassan et al., 2000; Hassan et al., 1989; Russell et al., 2002; DeLima et al., 2004; Madden et al., 2007; Andersson et al., 2008).

In further embodiments, azole compound may be used in the novelformulations and methods for improved aqueous solubility and stability,such as itraconazole (Itra) and posaconazole (Posa). The antifungalazole agents itraconazole (Itra) and posaconazole (Posa), that belong tothe general class of agents commonly referred to as tri-azole compounds,have earned an impressive reputation for their efficacy against bothyeast and various molds. The introduction of such azoles in clinicalmedicine has greatly improved the control of systemic fungal infectionsin both HIV- and non-HIV-infected immunocompromised individuals. Thesecompounds are active against a variety of fungal infections such asaspergillosis, blastomycosis, histoplasmosis, and candidiasis, as wellas fungal infections localized to the toenails and fingernails(onychomycosis), and to infections of the skin and reproductive tract(primarily referred to as “vaginal yeast infections”). They are alsoused for empirically and preemptively treating immunocompromisedpatients with fever and low white blood cell counts who are likely todevelop a fungal infection after radio- or chemotherapy for malignantdisease. The usual recommended dose varies between the different membersof the azole family in a single dose or two to three divided dailydoses. Capsules should be taken with a full meal becauselipid-containing food improves absorption.

Itra, as a representative example of orally administered antifungalagent(s)/(tri)-azoles, has previously been extensively investigated inhumans and domestic animals (Baddley et al., 2009; Campo et al., 2010;Chen et al., 2010; Dutkiewicz and Hage, 2010; Evans, 2010; Glockner andKarthaus, 2010; Hicheri et al., 2010; Hsu et al., 2010; Ito et al.,2010; Jang et al., 2010; Kim et al., 2010; Lehrnbecher et al., 2010;Lewis and Kontoyiannis, 2009; Lortholary et al., 2010; Pappas et al.,2010; Person et al., 2010; Singh et al., 2006; Torres et al., 2005;Ullmann et al., 2007; Vehreschild et al., 2010; Walsh et al., 2010;Wingard et al., 2010; Winston et al., 2010; Greer, 2007; Carrillo-Munozet al., 2005; Dodds-Ashley and Alexander, 2005; Groll and Walsh, 2006;Notheis et al., 2006; Courtney et al., 2003; Zhou et al., 1998; Bootheet al., 1997; Davis et al., 2005; Willems et al., 2001); the(se) drug(s)has (have) well documented anti-infectious properties in both clinicaland experimental settings. However, prior to the present invention, (an)acceptable parenteral formulation(s) of solubilized Itra, Posa and othermembers of this diverse family of chemicals either referred to astri-azoles, or simply azole compounds, have not been consistentlyavailable, but parenteral administration has been accomplished byallowing the use of microcrystalline suspensions of these azoles. Thevariable and somewhat unreliable stability of such formulations havegiven varying, unpredictable results. Thus, voriconazole is commerciallyavailable as such a formulation, while Itra was voluntarily withdrawnfrom the U.S. market by its manufacturer, and Posa remains unavailabledespite repeated attempts to provide a clinically useful parenteralformulation.

Truly solubilized, parenteral formulations of Itra and Posa would beuseful as treatment of systemic infectious disorders inimmunocompromised patients who for a multitude of reasons are unable toconsistently take oral preparations, such as e.g. commonly experiencedafter (intensive) conventional chemotherapy for acute leukemia and othermalignant diseases, and after (allogeneic) hematopoietic stem celltransplantation, where in the early post-transplant phase drug-relatednausea, vomiting and diarrhea as well as administration of concomitantmedications may impair oral drug bioavailability while later on theoccurrence of intestinal graft-vs-host disease and its therapy mayresult in a similar situation. In such patients parenteral drugadministration gives complete control of systemic drugdelivery/pharmacokinetics of the delivered agent with an accuracy simplynot attainable with an oral formulation (Benet and Sheiner, 1985).Unfortunately, Itra is a poorly water-soluble agent with exceedingly lowsolubility in physiologically acceptable aqueous solvents/infusionfluids that would be compatible with human administration. Prior to theinvention, the only currently available administration form is oralpreparations (capsules and an oral suspension), while a previouslyavailable microcrystalline suspension for IV use was withdrawn by itssupplier shortly after FDA-approval due to its unpredictablepharmaceutical behavior. To the inventors' knowledge a truly solubilizedform of Itra has never been available, but only a colloidal, ormicrocrystalline suspension in hydroxypropyl-beta-cyclodextrin (Willemset al., 2001).

As shown in the Examples, when Itraconazole (Itra) and Posaconazole(Posa) were injected at a dose of 5 mg/kg BW in mice after dissolvingand diluting in an analogous fashion (Stability data for the stock- andfinal use-formulation shown in FIGS. 9 and 10, from ten minutes to atleast 2 hours after drug injection the plasma Posa concentrationsremained in the 3-5 μg/mL range, and Itra was also detected at more than0.5 μg/mL over the same time interval. These concentration ranges aresimilar to what is expected when administering an oral dose equivalentof each drug in a clinical situation (Woestenborghs et al., 1987;Notheis et al., 2006; Courtney et al., 2003; Jang et al., 2010), andthese concentrations clearly exceed the minimum inhibitoryconcentrations of prototype mold strains that are pathogenic toimmunocompromised humans.

A variety of biological and chemical methods were used to demonstratethat preferred Bu and azole formulations are stable at approximately 5mg/mL for several weeks at RT. As shown in the Examples, one suchformulation (Bu/VE-acetone/PEG) is stable for greater than 40 or even 60days, and it retains full cytotoxic activity when assayed in vitroagainst human leukemic cell lines. Commercially available Bu wasdissolved in DMSO and used as a reference solvent system (“D”, or“DMSO”) for the in vitro cytotoxicity assay. The DMA-Bu formulation wasincluded in some experiments as a positive control in parallel; due tothe added synergistic cytotoxic effects of DMA the latter formulationwas clearly more toxic in the tested human cell lines. The novelBu/VE-acetone/PEG/dextrose vehicle is in itself virtually nontoxic asassayed in the hemolysis assay. Finally, one of the novel formulationswas used to show that cytocidal Bu concentrations/antifungal azoleconcentrations are maintained for several hours in a murine model afterIV injection of 10 mg/kg BW and 5 mg/kg BW, respectively.

Although a preferred embodiment of the invention uses acetone and PEG,with D5W as the secondary diluent, other solvent vehicles/diluents thatare non-toxic and safe for human administration may be used. No seriousclinical adverse effects have been experienced from the use of thesediluents. As alternatives to acetone alone, one could also use acidifiedacetone to allow protonation of reactive groups in the pharmacologicallyactive hydrophobic agent to further enhance its solubility andcomplex-formation with PEG, likely due to improved electrostaticattraction between the solute and PEG. Alternatively, it is possible touse other volatile organic solvents, such as chloroform by itself oracidified chloroform. For example, the acetone comprises between 1 and100% of the first solvent and PEG is the preferred second stock solvent;as an alternative, acetone comprises between 95 and 100% of the firstsolvent and a protonating agent, such as an acid or an alcohol,comprises between 0 and 5% of the first solvent.

Useful infusion fluids include, but are not limited to, normal salineand dextrose in water, or dextrose in water mixed with a protonatingagent such as an acid (Martin and Matzke, 1982), or dextrose in wateradmixed with a small amount of an amphilic solvent such as PEG tofurther decrease the risk of precipitation when the terminal aqueousdiluent is added to the drug stock-formulation. Alternatively, theinfusion fluid may be a lipid-based emulsion infusion fluid such asthose used for parenteral nutrition (Fortner et al., 1975). Prior todilution with the infusion fluid, the composition may comprise between 1and 20 mg/mL of a lipophilic agents such as Bu and, more preferably,comprises between 1 and 5 mg/mL of a lipophilic agents such as Bu.Preferably, the undiluted stock composition is stable for more than 30days at RT. The clinical use of normal saline (NS), dextrose in water(5-10%), and aqueous lipid emulsions are established, routine means tocorrect fluid and electrolyte balance and to supply parenteralnutrition. Normal saline and dextrose in water, are also extensivelyused to dilute various medications for IV use. The aqueous lipidemulsion has not yet found widespread use as a pharmaceutical diluent,but this use has been suggested (Fortner et al., 1975). Similarly, theintravenous administration of (hydrochloric) acid has been used for(rapid) correction of serious metabolic acidosis, but it has not beendescribed as a means to enhance protonation to maintain electrostaticattraction forces between a pharmaceutically active agent and disparatehydrophobic/amphiphilic solvents prior to administration in mammals(Martin and Matzke, 1982). In a particularly preferred embodiment, thesecondary diluent is 5-10% dextrose in water and the compositioncomprises between 0.5 and 2.0 mg/mL of Bu after dilution in thesecondary diluent. This diluted composition is stable for at least 12-15hours at RT.

The novel solutions of the invention are not limited to Bu, but may alsobe used to facilitate parenteral administration of other hydrophobic,and hard-to-solubilize, aka water-insoluble, drugs. As noted, suchagents include, but are not limited to, cytotoxic agents such asderivatives of epipodophyllotoxin, taxanes, Bleomycin, anthracyclines,as well as platinum compounds and camptothecin. They also includeantibiotics, such as the poorly water-soluble polyenes and azoles (e.g.,Amphotericin B and Natamycin, as well as the antifungal azolesincluding, but not limited to, itraconazole and posaconazole) as well asantibacterial agents, (e.g., polymyxin B), anti-viral agents andtranquilizing/anesthetic drugs such as benzodiazepines, Propofol andanti-psychotic agents.

Additional examples of lipophilic agents that can be used in accordancewith the present invention include, but are not limited to, lipophilicactive compounds or a salt, isomer, ester, ether or other derivativethereof selected from:

(i) acetylcholinesterase inhibitors selected from donepezil, tacrine,pyridostigmine;

(ii) analgesics and nonsteroidal antiinflammatory agents (NSAIA)selected from aloxiprin, auranofin, azapropazone, benorylate, capsaicin,celecoxib, diclofenac, diflunisal, etodolac, fenbufen, fenoprofencalcium, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac,leflunomide, meclofenamic acid, mefenamic acid, nabumetone, naproxen,oxaprozin, oxyphenbutazone, phenylbutazone, piroxicam, rofecoxib,sulindac, tetrahydrocannabinol, tramadol and tromethamine,

(iii) anthelminthics selected from albendazole, bepheniumhydroxynaphthoate, cambendazole, dichlorophen, fenbendazole, ivermectin,mebendazole, oxamniquine, oxfendazole, oxantel embonate, praziquantel,pyrantel embonate and thiabendazole;

(iv) antiacne agents such as isotretinoin and tretinoin;

(v) antianginal agents selected from amyl nitrate, glyceryl trinitrate(nitroglycerin), isosorbide dinitrate, isosorbide mononitrate,pentaerythritol tetranitrate, and ubidecarenone (coenzyme Q10);

(vi) antiarrhythmic agents selected from amiodarone HCl, digoxin,disopyramide, flecamide acetate and quinidine sulfate;

(vii) anti-asthma agents selected from zileuton, zafirlukast,terbutaline sulfate, montelukast, and albuterol;

(viii) antibacterial agents, including antibiotics, selected fromalatrofloxacin, azithromycin, aztreonam, baclofen, benzathinepenicillin, cefixime, cefuraxime axetil, cinoxacin, ciprofloxacin HCl,clarithromycin, clofazimine, cloxacillin, demeclocycline, dirithromycin,doxycycline, erythromycin, ethionamide, furazolidone, grepafloxacin,imipenem, levofloxacin, lorefloxacin, moxifloxacin HCl, nalidixic acid,nitrofurantoin, norfloxacin, ofloxacin, phenoxymethyl penicillin,rifabutine, rifampicin, rifapentine, sparfloxacin, spiramycin,sulphabenzamide, sulphadoxine, sulphamerazine, sulphacetamide,sulphadiazine, sulphafurazole, sulpha-methoxazole, sulphapyridine,tetracycline, trimethoprim, trovafloxacin, and vancomycin;

(ix) anti-benign prostate hypertrophy (BPH) agents selected fromalfuzosin, doxazosin, phenoxybenzamine, prazosin, terazosin andtamulosin;

(x) anticancer agents and immunosuppressants selected from abarelix,aldesleukin, alemtuzumab, alitretinoin, all-trans retinoic acid (ATRA),altretamine, amifostine, aminoglutethimide, amsacrine, anastrozole,arsenic trioxide, asparaginase, azacitidine, azathioprine, BCG Live,bevacizumab (avastin), bexarotene, bicalutamide, bisantrene, bleomycin,bortezomib, busulfan, calusterone, camptothecin, capecitabine,carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cisplatin,cladribine, clofarabine, cyclophosphamide, cyclosporin, cytarabine,dacarbazine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin,dexrazoxane, docetaxel, doxorubicin (neutral), doxorubicin HCl,dromostanolone propionate, ellipticine, enlimomab, estramustine,epirubicin, epoetin alfa, erlotinib, estramustine, etoposide,exemestane, filgrastim, floxuridine fludarabine, fulvestrant, gefitinib,gemcitabine, gemtuzumab, goserelin acetate, histrelin acetate,hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib mesylate,interferon alfa-2a, interferon alfa-2b, irinotecan, lenalidomide,letrozole, leucovorin, leuprolide acetate, levamisole, lomustine,megestrol acetate, melphalan, mercaptopurine, mesna, methotrexate,methoxsalen, mitomycin C, mitotane, mitoxantrone, mofetil mycophenolate,nandrolone, nelarabine, nilutamide, nofetumomab, oprelvekin,oxaliplatin, paclitaxel, palifermin, pamidronate, pegademase,peg-asparaginase, peg-filgrastim, pemetrexed disodium, pentostatin,pipobroman, plicamycin, porfimer sodium, procarbazine, quinacrine,rasburicase, rituximab, sargramostim, sirolimus, sorafenib,streptozocin, sunitinib maleate, tacrolimus, tamoxifen citrate,temozolomide, teniposide, testolactone, thioguanine, thiotepa,topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracilmustard, valrubicin, vinblastine, vincristine, vinorelbine, zoledronate,and zoledronic acid;

(xi) anticoagulants selected from cilostazol, clopidogrel, dicumarol,dipyridamole, nicoumalone, oprelvekin, phenindione, ticlopidine, andtirofiban;

(xii) antidepressants selected from amoxapine, bupropion, citalopram,clomipramine, fluoxetine HCl, maprotiline HCl, mianserin HCl,nortriptyline HCl, paroxetine HCl, sertraline HCl, trazodone HCl,trimipramine maleate, and venlafaxine HCl;

(xiii) antidiabetics selected from acetohexamide, chlorpropamide,glibenclamide, gliclazide, glipizide, glimepiride, glyburide, miglitol,pioglitazone, repaglinide, rosiglitazone, tolazamide, tolbutamide andtroglitazone;

(xiv) antiepileptics selected from beclamide, carbamazepine, clonazepam,thotoin, felbamate, fosphenyloin sodium, lamotrigine, methoin,methsuximide, methylphenobarbitone, oxcarbazepine, paramethadione,phenacemide, phenol barbitone, phenyloin, phensuximide, primidone,sulthiame, tiagabine HCl, topiramate, valproic acid, and vigabatrin;

(xv) antifungal agents selected from amphotericin, butenafine HCl,butoconazole nitrate, clotrimazole, econazole nitrate, fluconazole,flucytosine, griseofulvin, Itraconazole, ketoconazole, miconazole,natamycin, nystatin, sulconazole nitrate, oxiconazole, Posaconazole,terbinafine HCl, terconazole, tioconazole and undecenoic acid;

(xvi) antigout agents selected from allopurinol, probenecid andsulphinpyrazone;

(xvii) antihypertensive agents selected from amlodipine, benidipine,benezepril, candesartan, captopril, darodipine, dilitazem HCl,diazoxide, doxazosin HCl, enalapril, eposartan, losartan mesylate,felodipine, fenoldopam, fosenopril, guanabenz acetate, irbesartan,isradipine, lisinopril, minoxidil, nicardipine HCl, nifedipine,nimodipine, nisoldipine, phenoxybenzamine HCl, prazosin HCl, quinapril,reserpine, terazosin HCl, telmisartan, and valsartan;

(xviii) antimalarial agents selected from amodiaquine, chloroquine,chlorproguanil HCl, halofantrine HCl, mefloquine HCl, proguanil HCl,pyrimethamine and quinine sulfate;

(xix) antimigraine agents selected from dihydroergotamine mesylate,ergotamine tartrate, frovatriptan, methysergide maleate, naratriptanHCl, pizotifen maleate, rizatriptan benzoate, sumatriptan succinate, andzolmitriptan;

(xx) antimuscarinic agents selected from atropine, benzhexol HCl,biperiden, ethopropazine HCl, hyoscyamine, mepenzolate bromide,oxyphencyclimine HCl and tropic amide

(xxi) antiparkinsonian agents selected from bromocriptine mesylate,lysuride maleate, pramipexole, ropinirole HCl, and tolcapone;

(xxii) antiprotozoal agents selected from atovaquone, benznidazole,clioquinol, decoquinate, diiodohydroxyquinoline, diloxamide furoate,dinitolmide, furazolidone, metronidazole, nimorazole, nitrofurazone,ornidazole and tinidazole;

(xxiii) antithyroid agents selected from carbimazole andpropylthiouracil;

(xxiv) antitussive agent such as benzonatate;

(xxv) antiviral agents selected from abacavir, amprenavir, delavirdine,efavirenz, indinavir, lamivudine, nelfinavir, nevirapine, ritonavir,saquinavir, and stavudine;

(xxvi) anxiolytics, sedatives, hypnotics and neuroleptics selected fromalprazolam, amylobarbitone, barbitone, bentazepam, bromazepam,bromperidol, brotizolam, butobarbitone, carbromal, chlordiazepoxide,chlormethiazole, chlorpromazine, chlorprothixene, clonazepam, clobazam,clotiazepam, clozapine, diazepam, droperidol, ethinamate, flunanisone,flunitrazepam, triflupromazine, flupenthixol decanoate, fluphenthixoldecanoate, flurazepam, gabapentin, haloperidol, lorazepam, lormetazepam,medazepam, meprobamate, mesoridazine, methaqualone, methylphenidate,midazolam, molindone, nitrazepam, olanzapine, oxazepam, pentobarbitone,perphenazine pimozide, prochlorperazine, propofol, pseudoephedrine,quetiapine, risperidone, sertindole, sulpiride, temazepam, thioridazine,triazolam, zolpidem, and zopiclone;

(xxvii) beta.-blockers selected from acebutolol, alprenolol, atenolol,labetalol, metoprolol, nadolol, oxprenolol, pindolol and propranolol;

(xxviii) cardiac inotropic agents selected from anrinone, digitoxin,digoxin, enoximone, lanatoside C and medigoxin;

(xxix) corticosteroids selected from beclomethasone, betamethasone,budesonide, cortisone acetate, desoxymethasone, dexamethasone,fludrocortisone acetate, flunisolide, fluocortolone, fluticasonepropionate, hydrocortisone, methylprednisolone, prednisolone, prednisoneand triamcinolone;

(xxx) diuretics selected from acetazolamide, amiloride,bendroflumethiazide, bumetanide, chlorothiazide, chlorthalidone,ethacrynic acid, frusemide, metolazone, spironolactone and triamterene;

(xxxi) gastrointestinal agents selected from bisacodyl, cimetidine,cisapride, diphenoxylate HCl, domperidone, famotidine, lanosprazole,loperamide, mesalazine, nizatidine, omeprazole, ondansetron HCl,pantoprazole, rabeprazole sodium, ranitidine HCl and sulphasalazine;

(xxxii) histamine H1- and H2-receptor antagonists selected fromacrivastine, astemizole, chlorpheniramine, cinnarizine, cetrizine,clemastine fumarate, cyclizine, cyproheptadine HCl, dexchlorpheniramine,dimenhydrinate, fexofenadine, flunarizine HCl, loratadine, meclizineHCl, oxatomide, and terfenadine;

(xxxiii) keratolytic agents selected from acetretin, calciprotriene,calcifediol, calcitriol, cholecalciferol, ergocalciferol, etretinate,retinoids, targretin, and tazarotene;

(xxxiv) lipid regulating/hypolipidemic agents selected fromatorvastatin, bezafibrate, cerivastatin, ciprofibrate, clofibrate,fenofibrate, fluvastatin, gemfibrozil, hesperetin, lovastatin,pravastatin, probucol, and simvastatin;

(xxxv) muscle relaxants selected from cyclobenzaprine, dantrolene sodiumand tizanidine HCl;

(xxxvi) opioid analgesics selected from codeine, dextropropoxyphene,diamorphine, dihydrocodeine, fentanyl, meptazinol, methadone, morphine,nalbuphine and pentazocine;

(xxxvii) sex hormones selected from clomiphene citrate, cortisoneacetate, danazol, dehydroepiandrosterone, ethynylestradiol, finasteride,fludrocortisone, fluoxymesterone, medroxyprogesterone acetate, megestrolacetate, mestranol, methyltestosterone, mifepristone, norethisterone,norgestrel, oestradiol, conjugated estrogens, progesterone, rimexolone,stanozolol, stilbestrol, testosterone and tibolone;

(xxxviii) stimulants selected from amphetamine, dexamphetamine,dexfenfluramine, fenfluramine and mazindol;

(xxxix) nutraceutical agents selected from calcitriol, carotenes,chrysin, dihydrotachysterol, flavonoids, hesperitin, jasmonates, lipoicacid, lutein, lycopene, essential fatty acids, non-essential fattyacids, naringenin, phytonadiol, quercetin, vitamins including vitamin A,vitamin B2, vitamin D and derivatives, vitamin E, and vitamin K,coenzyme Q10 (ubiquinone), plant extracts, and minerals.

Analgesics may be used in certain aspects of the invention. An analgesic(also known as a painkiller) is any member of the group of drugs used torelieve pain (achieve analgesia). The word analgesic derives from Greekan- (“without”) and algos (“pain”).

Analgesic drugs act in various ways on the peripheral and centralnervous systems; they include paracetamol (para-acetylaminophenol, alsoknown in the US as acetaminophen), the non-steroidal anti-inflammatorydrugs (NSAIDs) such as the salicylates, and opioid drugs such asmorphine and opium. They are distinct from anesthetics, which reversiblyeliminate sensation.

Nonsteroidal anti-inflammatory drugs, usually abbreviated to NSAIDs orNAIDs, but also referred to as nonsteroidal anti-inflammatoryagents/analgesics (NSAIAs) or nonsteroidal anti-inflammatory medicines(NSAIMs), are drugs with analgesic and antipyretic (fever-reducing)effects and which have, in higher doses, anti-inflammatory effects.NSAIDs are generally indicated for the symptomatic relief of thefollowing conditions: Rheumatoid arthritis, Osteoarthritis, Inflammatoryarthropathies (e.g. ankylosing spondylitis, psoriatic arthritis,Reiter's syndrome), Acute gout, Dysmenorrhoea (menstrual pain),Metastatic bone pain, Headache and migraine, Postoperative painMild-to-moderate pain due to inflammation and tissue injury, Pyrexia(fever), Ileus, Renal colic, or neonate infants whose ductus arteriosusis not closed within 24 hours of birth.

COX-2 selective inhibitor is a form of non-steroidal anti-inflammatorydrug (NSAID) that directly targets COX-2, an enzyme responsible forinflammation and pain. Selectivity for COX-2 reduces the risk of pepticulceration, and is the main feature of celecoxib, rofecoxib and othermembers of this drug class. COX-2 selectivity does not seem to reduceother adverse effects of NSAIDs (most notably an increased risk of renalfailure), and some results have shown an increase in the risk for heartattack, thrombosis and stroke by a relative increase in thromboxane.Rofecoxib is one example.

Flupirtine is a centrally acting K+ channel opener with weak NMDAantagonist properties. It is used in Europe for moderate to strong painand migraine and its muscle relaxant properties. It has noanticholinergic properties and is believed be devoid of any activity ondopamine, serotonin or histamin receptors. It is not addictive andtolerance does not develop.

In patients with chronic or neuropathic pain, various other substancesmay have analgesic properties. Tricyclic antidepressants, especiallyamitriptyline, have been shown to improve pain in what appears to be acentral manner. Nefopam is used in Europe for pain relief withconcurrent opioids. The exact mechanism of carbamazepine, gabapentin andpregabalin is similarly unclear, but these anticonvulsants are used totreat neuropathic pain with differing degrees of success.Anticonvulsants are most commonly used for neuropathic pain as theirmechanism of action tends to inhibit pain sensation.

Antidepressants may be used in certain aspects of the invention.Tricyclic antidepressants (TCAs) are heterocyclic chemical compoundsused primarily as antidepressants. The TCAs were first discovered in theearly 1950s and were subsequently introduced later in the decade; Theyare named after their chemical structure, which contains three rings ofatoms. The tetracyclic antidepressants (TeCAs), which contain four ringsof atoms, are a closely related group of antidepressant compounds.

The TCAs include the following agents which are predominantly serotoninand/or norepinephrine reuptake inhibitors: Amitriptyline (Elavil,Tryptizol, Laroxyl), Amitriptylinoxide (Amioxid, Ambivalon, Equilibrin),Butriptyline (Evadyne), Clomipramine (Anafranil), Demexiptiline(Deparon, Tinoran), Desipramine (Norpramin, Pertofrane), Dibenzepin(Noveril, Victoril), Dimetacrine (Istonil, Istonyl, Miroistonil),Dosulepin/Dothiepin (Prothiaden), Doxepin (Adapin, Sinequan), Imipramine(Tofranil, Janimine, Praminil), Imipraminoxide (Imiprex, Elepsin),Lofepramine (Lomont, Gamanil), Melitracen (Deanxit, Dixeran, Melixeran,Trausabun), Metapramine (Timaxel), Nitroxazepine (Sintamil),Nortriptyline (Pamelor, Aventyl), Noxiptiline (Agedal, Elronon,Nogedal), Pipofezine (Azafen/Azaphen), Propizepine (Depres sin, Vagran),Protriptyline (Vivactil), Quinupramine (Kevopril, Kinupril, Adeprim,Quinuprine)

As well as the following atypical compounds: Amineptine (Survector,Maneon, Directim)—Norepinephrine-dopamine reuptake inhibitor; Iprindole(Prondol, Galatur, Tetran)—5-HT2 receptor antagonist; Opipramol(Insidon, Pramolan, Ensidon, Oprimol)—σ receptor agonist; Tianeptine(Stablon, Coaxil, Tatinol)—Selective serotonin reuptake enhancer;Trimipramine (Surmontil)—5-HT2 receptor antagonist

In recent times, the TCAs have been largely replaced in clinical use inmost parts of the world by newer antidepressants such as the selectiveserotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrinereuptake inhibitors (SNRIs), which typically have more favourableside-effects profiles, though they are still sometimes prescribed forcertain indications.

Selective serotonin reuptake inhibitors or serotonin-specific reuptakeinhibitor. (SSRIs) are a class of compounds typically used asantidepressants in the treatment of depression, anxiety disorders, andsome personality disorders. They are also typically effective and usedin treating some cases of insomnia. The main indication for SSRIs isclinical depression. SSRIs are frequently prescribed for anxietydisorders, such as social anxiety, panic disorders, obsessive-compulsivedisorder (OCD), eating disorders, chronic pain and occasionally, forposttraumatic stress disorder (PTSD). Though not specifically indicatedby the manufacturers, they are sometimes prescribed to treat irritablebowel syndrome (IBS), Lichen simplex chronicus and prematureejaculation. All SSRIs are approved in the U.S. for use with psychiatricdisorders as outlined in the Diagnostic and Statistical Manual of MentalDisorders (DSM IV).

SSRIs are believed to increase the extracellular level of theneurotransmitter serotonin by inhibiting its reuptake into thepresynaptic cell, increasing the level of serotonin in the synapticcleft available to bind to the postsynaptic receptor. They have varyingdegrees of selectivity for the other monoamine transporters, with pureSSRIs having only weak affinity for the noradrenaline and dopaminetransporter.

SSRI Drugs include (trade names in parentheses): citalopram (Celexa,Cipramil, Cipram, Dalsan, Recital, Emocal, Sepram, Seropram, Citox,Cital); dapoxetine (Priligy); escitalopram (Lexapro, Cipralex, Seroplex,Esertia); fluoxetine (Prozac, Fontex, Seromex, Seronil, Sarafem, Ladose,Motivest, Flutop, Fluctin (EUR), Fluox (NZ), Depress (UZB), Lovan (AUS);fluvoxamine (Luvox, Fevarin, Faverin, Dumyrox, Favoxil, Movox);indalpine (Upstene) (discontinued); paroxetine (Paxil, Seroxat,Sereupin, Aropax, Deroxat, Divarius, Rexetin, Xetanor, Paroxat,Loxamine, Deparoc); sertraline (Zoloft, Lustral, Serlain, Asentra);vilazodone (Viibyrd); zimelidine (Zelmid, Normud) (discontinued).

Serotonin-norepinephrine reuptake inhibitors (SNRIs) are a class ofantidepressant drugs used in the treatment of major depression and othermood disorders. They are sometimes also used to treat anxiety disorders,obsessive-compulsive disorder (OCD), attention deficit hyperactivitydisorder (ADHD), chronic neuropathic pain, fibromyalgia syndrome (FMS),and for the relief of menopausal symptoms.

SNRIs act upon and increase the levels of two neurotransmitters in thebrain that are known to play an important part in mood, these beingserotonin and norepinephrine. This can be contrasted with the morewidely-used selective serotonin reuptake inhibitors (SSRIs) which onlyact on serotonin.

Examples of SNRIs include:

Venlafaxine (Effexor)—The first and most commonly used SNRI. It wasintroduced by Wyeth in 1994. The reuptake effects of venlafaxine aredose dependent. At low doses (<150 mg/day) it acts only on serotonergictransmission. At moderate doses (>150 mg/day) it acts on serotonergicand noradrenergic systems, whereas at high doses (>300 mg/day) it alsoaffects dopaminergic neurotransmission.

Desvenlafaxine (Pristiq)—The active metabolite of venlafaxine. It isbelieved to work in a similar manner, though some evidence suggestslower response rates compared to venlafaxine and duloxetine. It wasintroduced by Wyeth in May 2008.

Duloxetine (Cymbalta, Yentreve)—By Eli Lilly and Company, has beenapproved for the treatment of depression and neuropathic pain in August2004. Duloxetine is contraindicated in patients with heavy alcohol useor chronic liver disease, as duloxetine can increase the levels ofcertain liver enzymes which can lead to acute hepatitis or otherdiseases in certain at risk patients. Currently, the risk of liverdamage appears only to be for patients already at risk, unlike theantidepressant nefazodone which, though rare, can spontaneously causeliver failure in healthy patients. Duloxetine is also approved for MajorDepressive Disorder (MDD), Generalized Anxiety Disorder (GAD), chronicmusculoskeletal pain, including chronic osteoarthritis pain and chroniclow back pain (as of October, 2010), and is one of the only threemedicines approved by the FDA for Fibromyalgia.

Milnacipran (Dalcipran, Ixel, Savella)—Shown to be significantlyeffective in the treatment of depression and fibromyalgia. The Food andDrug Administration (FDA) approved milnacipran for treatment offibromyalgia in the United States of America in January 2009, however itis currently not approved for depression in that country. Milnacipranhas been commercially available in Europe and Asia for several years.

Levomilnacipran (F2695)—The levo-isomer of milnacipran. Underdevelopment for the treatment of depression in the United States andCanada.

Sibutramine (Meridia, Reductil)—An SNRI, which, instead of beingdeveloped for the treatment of depression, was widely marketed as anappetite suppressant for weight loss purposes.

Bicifadine (DOV-220,075)—By DOV Pharmaceutical, potently inhibits thereuptake of serotonin and norepinephrine (and dopamine to a lesserextent), but rather than being developed for the already crowdedantidepressant market, it is being researched as a non-opioid, non-NSAIDanalgesic.

SEP-227162—An SNRI under development by Sepracor for the treatment ofdepression.

LY 2216684—An SNRI under development by Eli Lilly for the treatment ofdepression.

Antipsychotics may be used in certain aspects of the invention. Anantipsychotic (or neuroleptic) is a tranquilizing psychiatric medicationprimarily used to manage psychosis (including delusions orhallucinations, as well as disordered thought), particularly inschizophrenia and bipolar disorder. A first generation ofantipsychotics, known as typical antipsychotics, was discovered in the1950s. Most of the drugs in the second generation, known as atypicalantipsychotics, have been developed more recently, although the firstatypical antipsychotic, clozapine, was discovered in the 1950s andintroduced clinically in the 1970s. Both generations of medication tendto block receptors in the brain's dopamine pathways, but antipsychoticdrugs encompass a wide range of receptor targets.

Common conditions with which antipsychotics might be used includeschizophrenia, bipolar disorder and delusional disorder. Antipsychoticsmight also be used to counter psychosis associated with a wide range ofother diagnoses, such as psychotic depression. However, not all symptomsrequire heavy medication and hallucinations and delusions should only betreated if they distress the patient or produce dangerous behaviors.

In addition, “antipsychotics” are increasingly used to treatnon-psychotic disorders. For example, they are sometimes used off-labelto manage aspects of Tourette syndrome or autism spectrum disorders.They have multiple off-label uses as an augmentation agent (i.e. inaddition to another medication), for example in “treatment-resistant”depression or OCD. Despite the name, the off-label use of“antipsychotics” is said to involve deploying them as antidepressants,anti-anxiety drugs, mood stabilizers, cognitive enhancers,anti-aggressive, anti-impulsive, anti-suicidal and hypnotic (sleep)medications.

Antipsychotics have also been increasingly used off-label in cases ofdementia in older people, and for various disorders and difficulties inchildren and teenagers. A survey of children with pervasivedevelopmental disorder found that 16.5% were taking an antipsychoticdrug, most commonly to alleviate mood and behavioral disturbancescharacterized by irritability, aggression, and agitation. Recently,risperidone was approved by the US FDA for the treatment of irritabilityin children and adolescents with autism.

Antipsychotics may include first generation antipsychotics (typicalantipsychotic), Butyrophenones, Phenothiazines, Thioxanthenes, secondgeneration antipsychotics (atypical antipsychotic), third generationantipsychotics, Cannabidiol (CBD), etc.

Antipsychotics are sometimes used as part of compulsory treatment viainpatient (hospital) commitment or outpatient commitment. This mayinvolve various methods to persuade a person to take the medication, oractual physical force. Administration may rely on an injectable form ofthe drug rather than tablets. The injection may be of a long-lastingtype known as a depot injection, usually applied at the top of thebuttocks. Those that are available in injectable form are haloperidol,olanzapine, and ziprasidone while those available as depot arehaloperidol, flupenthixol, clopenthixol, and risperidone.

The atypical antipsychotics (AAP) (also known as second generationantipsychotics) are a group of antipsychotic tranquilizing drugs used totreat psychiatric conditions. Some atypical antipsychotics are FDAapproved for use in the treatment of schizophrenia. Some carry FDAapproved indications for acute mania, bipolar depression, psychoticagitation, bipolar maintenance, and other indications. The atypicalantipsychotics may include: Amisulpride (Solian), Aripiprazole(Abilify), Asenapine (Saphris), Blonanserin (Lonasen), Clotiapine(Entumine), Clozapine (Clozaril), Iloperidone (Fanapt), Lurasidone(Latuda), Mosapramine (Cremin), Olanzapine (Zyprexa), Paliperidone(Invega), Perospirone (Lullan), Quepin (Specifar), Quetiapine(Seroquel), Remoxipride (Roxiam), Risperidone (Risperdal), Sertindole(Serdolect), Sulpiride (Sulpirid, Eglonyl), Ziprasidone (Geodon,Zeldox), Zotepine (Nipolept), Bifeprunox (DU-127,090), Pimavanserin(ACP-103), Vabicaserin (SCA-136). Third generation antipsychotics mayinclude Aripiprazole (Abilify) or partial agonists of dopamine.

Anesthetics may be used in certain aspects of the invention. Ananesthetic (or anaesthetic, see spelling differences) is a drug thatcauses anesthesia-reversible loss of sensation. They contrast withanalgesics (painkillers), which relieve pain without eliminatingsensation. These drugs are generally administered to facilitate surgery.A wide variety of drugs are used in modern anesthetic practice. Many arerarely used outside of anesthesia, although others are used commonly byall disciplines. Anesthetics are categorized in to two classes: generalanesthetics, which cause a reversible loss of consciousness, and localanesthetics, which cause a reversible loss of sensation for a limitedregion of the body while maintaining consciousness. Combinations ofanesthetics are sometimes used for their synergistic and additivetherapeutic effects, however, adverse effects may also be increased.

Local anesthetics are agents that prevent transmission of nerve impulseswithout causing unconsciousness. They act by binding to fast sodiumchannels from within (in an open state). Local anesthetics can be eitherester or amide based. Ester local anesthetics (e.g., procaine,amethocaine, cocaine) are generally unstable in solution andfast-acting, and allergic reactions are common. Non-limiting examples oflocal anesthetics may include procaine, amethocaine, cocaine, lidocaine(also known as Lignocaine), prilocaine, bupivacaine, levobupivacaine,ropivacaine, mepivacaine, and dibucaine. Amide local anesthetics (e.g.,lidocaine, prilocaine, bupivicaine, levobupivacaine, ropivacaine,mepivacaine and dibucaine) are generally heat-stable, with a long shelflife (around 2 years). They have a slower onset and longer half-lifethan ester anesthetics, and are usually racemic mixtures, with theexception of levobupivacaine (which is S(−)-bupivacaine) and ropivacaine(S(−)-ropivacaine). These agents are generally used within regional andepidural or spinal techniques, due to their longer duration of action,which provides adequate analgesia for surgery, labor, and symptomaticrelief. Only preservative-free local anesthetic agents may be injectedintrathecally.

A general anaesthetic (or anesthetic, see spelling differences) is adrug that brings about a reversible loss of consciousness. These drugsare generally administered by an anaesthesia provider to induce ormaintain general anaesthesia to facilitate surgery. The biologicalmechanism(s) of the action of general anaesthetics are not wellunderstood. Injection anaesthetics are used for induction andmaintenance of a state of unconsciousness. Anaesthetists prefer to useintravenous injections, as they are faster, generally less painful andmore reliable than intramuscular or subcutaneous injections. Among themost widely used drugs are: Propofol, Etomidate, Barbiturates such asmethohexital and thiopentone/thiopental, Benzodiazepine derivatives suchas midazolam, and Ketamine.

The compositions of the invention have a number of uses. The inventionmay include a method for treating a disease that is sensitive orresponsive to a lipophilic drug treatment comprising: parenterallyadministering a therapeutically effective amount of a parenteral drugcomposition as described above to a patient.

Diseases or conditions that may be treated include, but are not limitedto, cancer, acute and chronic leukemia, Hodgkin's and Non-Hodgkin'slymphoma, a myeloproliferative disorder, an autoimmune disease and asforming the basis of combination chemotherapy intended to prepare apatient for hematopoietic progenitor cell transplantation, infectiousdiseases, psychotic conditions or disease, or a need of symptom control.Preferably, the composition may be administered intravascularly, but asdetermined by specific clinical circumstances it may also be givenintrathecally, intracavitary (such as e.g. intraperitoneally, orintrapleurally) among other routes to treat locally advanced disease.After mixing with or suspending in a suitable ointment base, thecomposition may also be applied topically, such as in the treatment of aperipheral T-cell lymphoma or in the case of using the composition as abase solvent system for an anti-infectious composition suitable fortopical administration to accomplish a remedy for a localized infectiousor inflammatory ailment. The patient can be any animal. More preferably,the animal is a mammal, and most preferably, a human.

The term “therapeutically effective amount” as used in this applicationmeans that a sufficient amount of the composition is added to achievethe desired therapeutic effect or another effect, e.g., to transientlyachieve symptom control, or alter the patient's level of consciousness.The actual amount used will vary based on factors such as the type ofmedical condition, the age, sex, health, species and weight of thepatient, and the type of use and length of use, as well as other factorsknown to those skilled in the art.

Still another embodiment of the invention is directed to a method forparenterally administering a lipophilic agent to a patientcomprising: 1) providing a primary solvent vehicle in the form of anorganic solvent; 2) mixing the solubilized pharmaceutically activeagent, e.g., Bu, with a secondary hydrophobic/amphiphilic agent, such asPEG; 3) extracting the primary solvent, preferably under vacuum tocreate a microenvironment that is conducive to electrostatic binding ofthe pharmaceutically active agent to the polymer, PEG, thus keeping thepharmaceutically-active agent bound to or “dissolved” in PEG aftercompletion of the vacuum-extraction. This may prevent precipitation andthereby to produce a stock formulation; 4) mixing the stock formulationwith a secondary diluent to form an infusion fluid; and 5) administeringthe infusion fluid containing the pharmaceutically-active agent to thepatient. Preferably, the primary organic solvent is acetone. However, inaddition to acetone, many other organic solvents, such as chloroform anddiethyl-ether, may be used to form the primary diluent without departingfrom the spirit and scope of the invention.

As an example, Bu-based formulations may be useful in the treatment ofmalignancies and autoimmune diseases in man and animals. Certainhematologic malignancies, most notably the myeloid neoplasms, such asCML, AML, MDS, as well as Hodgkin's and Non-Hodgkin's lymphomas, may becontrolled with Bu-based therapy for prolonged time periods, especiallywhen utilized in pretransplant conditioning therapy. The nontoxic,pharmaceutically acceptable, water miscible, parenteral Bu-basedformulations eliminate the risk of treatment failure from unpredictableand erratic intestinal absorption and first-pass liverelimination/metabolism that to varying degrees characterizeadministration of the oral standard preparation, and it avoids theunpredictable and erratic toxicity of DMA used in the only currentlyavailable IV Bu-formulation. The potential benefits of the newparenteral formulation(s) not only include(s) fewer side effects thanthat experienced with oral drug, since intravascular administrationgives complete control of the drug's bioavailability andpharmacokinetics, but it avoids the unpredictable adverse synergisticinteraction(s) of Bu and DMA. It also safeguards against possibleunpredictable adverse interaction(s) between other concomitantlyadministered chemotherapeutic agents and DMA in the case of combinationchemotherapy. It should be noted that DMA, at the resulting finalconcentrations, contributed significant synergistic toxicity in at leastfour tested human cell lines, but this is conceivably also the case fornormal-organ toxicity in both human and animal settings as discussedabove.

Additional exemplary formulations of the invention may be useful in thetreatment of fungal, yeast and mold infections in mammals, particularlyCandida, Aspergillus or Mucorales infection. Certain infections, mostnotably those caused by Histoplasma Spp. and Aspergillus Spp. may besuccessfully controlled by Itra, and Posa has in addition been ofparticular value in treatment of mucormycosis in immunocompromisedpatients. The novel formulations, such as nontoxic, pharmaceuticallyacceptable, water-miscible, intravascular Itra or Posa formulations,eliminate the risk of treatment failure from unpredictable and erraticintestinal absorption and first-pass liver elimination/metabolism thatto varying degrees characterize administration of the oral standardpreparation(s). The potential benefits of using the intravascularadministration route/formulation is most evident in severely illpatients with an impaired ability to swallow and therefore unable tobenefit from oral nutrition such as for instance patients suffering fromoral and gastro-intestinal mucositis after radio- and/or chemotherapyfor neoplastic disease and those suffering from gastrointestinalgraft-vs-host disease after allogeneic stem cell transplantation where asimilar clinical conundrum exists. The benefits are also expected toinclude fewer clinical side effects than that experienced with thecorresponding oral drug formulation, since intravascular administrationgives complete control of the bioavailability with optimizedpharmacokinetics of the drugs and therefore minimizes the risk for sideeffects due to unwanted drug-drug interactions and treatment-failuresecondary to incomplete intestinal absorption as well as accidentaloverdosing in patients who have an unexpectedly high intestinalabsorption paired with a low metabolic drug clearance.

The novel formulations may also be used to investigate differentadministration schedules (e.g., prolonged IV infusions, and repeated IVdosing) to optimize treatment outcome for lipophilic drug-based therapy,such as for Bu-based therapy, because now that higher stable Buconcentrations (1-2 mg/mL) and extended stability (with Bu at least 15hours at RT in the final use-formulation, at 1 mg/mL) is obtained.Further, the invention makes it possible to investigate the benefits ofdifferent dose schedules of the lipophilic agents, such as busulfan orazole drugs against various systemic diseases, without the confoundingadverse effects from unpredictable intestinal drug absorption andhepatic first-pass effects that in an arbitrary fashion influence themetabolism of orally administered drugs.

The availability of a new parenteral preparation, especially one that isfree from DMA, is of particular interest when dose-intensive schedulesare contemplated, and in particular in the pediatric patient population,where side effects such as those exemplified by impaired growth andretardation of both physical and intellectual development may be morepronounced. In this specific situation a lack of solvent-system-relatedtoxicity linked together with absolute Bu bioavailability and precisepharmacokinetics are of utmost importance to ensure optimal patientsafety through control of the drug's clinical adverse effect profile,yet maximizing the chance for long-term disease control.

For infectious disease, the novel formulations and methods may obviatethe need to contend with the highly variable intestinal absorption thathas been reported between patients with different underlying diseases aswell as different age categories (Willems et al., 2001), and whether thepatient is fed or fasting (Dodds-Ashley, 2010; Willems et al., 2001; Vande Velde et al., 1996), and the novel formulations and methods alsoalleviate the need to worry about the “saturable” intestinal drugabsorption that has been described after Posa administration (Courtneyet al., 2003). The availability of a novel parenteral preparation may bealso important when more dose-intensive schedules are contemplated tocontrol overwhelming diseases or infections in severelyimmunocompromised patients, such as sino-pulmonary Aspergillosis andMucormycosis early after hematopoietic stem cell transplantation.Particularly, absolute drug bioavailability and predictablepharmacokinetics are of utmost importance to ensure the patient's safetythrough control of a drug's clinical side effects, while maximizing thechance for rapid control of a clearly life-threatening, rapidlyprogressive infectious complication in a very complex medical situation,where it is of crucial to rapidly establish control of the infection.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES Example 1—Busulfan and Azole Parenteral Formulations

This example demonstrates the successful design of stable formulationsof busulfan (Bu), using solvent vehicles that are nontoxic and suitablefor parenteral administration. The necessary solubility/stability wascalculated, and preparations were evaluated with high-pressure liquidchromatographic (HPLC) technique. The desired solubility and stabilityof Bu in various solvents relevant for parenteral, (as represented byintravascular or intrathecal or intracavitary) administration in humansand domestic animals was defined, and the solubility of Bu inphysiologically acceptable vehicles was determined.

Preparation of Prototype Solvent Vehicle and Primary Stock Solution.

A preferred Bu Vacuum-Extracted or Vacuum-Evaporated acetone(VE-acetone)/PEG/Bu (“primary stock solution”) as referenced in theseExamples was prepared as follows: 10 mg/mL of Bu was solubilized inacetone, mixed with PEG400 (1:2, v/v), then extracted at RT under vacuumfor at least 5 hours. The stability of Bu in VE-acetone/PEG at RT isgraphed in FIG. 1a . The stability of Bu in VE-acetone/PEG and dilutedin D5W at 0.5 mg/mL and 1 mg/mL is displayed in FIG. 1 b.

Calculation of Desired Solubility.

A relevant solubility range for Bu was calculated by extrapolation fromdoses known to have significant anti-tumor efficacy in man. Suchclinical studies have been conducted using oral Bu, as well as the IVDMA-Bu, the only FDA-approved parenteral Bu formulation. The utilized Buregimens typically prescribe a dose in the range of 2-10 mg orally oncedaily until clinical anti-tumor effect is obtained, or intermittentdoses of about 20-40 mg/m² of body surface area (BSA) as determined bywhite blood counts, in palliative therapy for CML or by repeatedadministration every 6-24 hours for 2-4 days when used in pre-HSCTconditioning therapy. The clinically most effective/optimal Bu-dose and-schedule are not known, but different approaches have been reported tobe successful. The dose-limiting toxicity with both the low-dose dailyschedules and the intermittent more intensive dose schedule are bonemarrow suppression. The administration of low daily doses over prolongedtime has correlated with a slow and insidious onset of bone marrowsuppression that is commonly considered safer and a preferred approachin palliative therapy.

The intermittent high-dose schedules of both oral and IV Bu that areextensively used in pretransplant conditioning therapy are typicallyconsidered dependent on hematopoietic progenitor cell support, sincesuch schedules are myeloablative. In pretransplant conditioning therapythe most commonly used oral Bu schedule is 1 mg/kg given every 6 hoursfor a total of 8-16 doses over 2-4 days. Thus, the parenteral DMA-Buformulation is recommended for pretransplant conditioning therapy at adose of 0.8 mg/kg given over 2 hours every 6 hours for 16 doses(Busulfex, 2009). The dose-limiting toxicity with the high-dosepretransplant regimens is typically mucositis, liver failure and about7-10% risk for generalized seizures (Santos et al., 1983; Tutschka etal., 1987; Ciurea et al., 2009; Blaise et al., 1992; Devergie et al.,1995; Hartman et al., 1998; Socie et al., 2001; Grochow et al., 1989;Grochow, 1993; Slattery et al., 1997; Dix et al., 1997; Marcus andGoldman, 1984; Santos, 1989; Vassal et al., 1990; Vassal et al., 1989;Martell et al., 1987; Sureda et al., 1989; Kashyap et al., 2002; Thallet al., 2004; Kim et al., 2005; Lee et al., 2005; Aggarwal et al., 2006;Dean et al., 2010; DeLeve et al., 2002; Russell et al., 2002; De Lima etal., 2004; Andersson et al., 2008). The risks connected with generalizedseizure activity as a side effect of high-dose Bu administration residesprimarily with (head-)trauma and bronchial aspiration of gastric contentduring the seizure episode. The seizures connected with busulfanadministration can be avoided through the prophylactic use ofbenzodiazepines or diphenylhydantoin (Grigg et al., 1989; Meloni et al.,1995; Chan et al., 2002; Hassan et al., 1993). The risk for seizures mayalso be influenced by the Bu administration schedule. Thus, a prolongedinfusion schedule provides extended tumor cell drug exposure, while atthe same time avoiding sudden high plasma peak concentrations that maybe involved in triggering more serious neurological side effects. Fromthese observations the inventors deduced that a stable final-useformulation with a Bu concentration that would allow prolonged infusionshould ideally contain between 1 and 2 mg/mL to limit the total volumeadministered over a short time in a once daily administration schedule.

Busulfan has a short terminal half-life in blood, approximately 2-4hours, and a prolonged infusion would extend drug exposure to themalignant tissues, yet decrease the plasma peak drug concentration thatmay be more closely associated with serious neurological side effects.

A solvent system was discovered that provides a use-formulation that isstable (>95%) at room temperature (RT) for long periods of time. FIG. 1ais a graph showing the stability of Bu at room temperature (RT) in thestock-formulation of VE-acetone/PEG (i.e. prototype solvent vehicle)containing Bu at approximately 5 mg/mL. Busulfan dissolved in a solventvehicle of VE-acetone/PEG, and further diluted to appropriateconcentrations with D5W or D10W with 10-20% PEG or with normal saline(NS) to 1 mg/mL is stable at RT for at least 15 hours. The results shownin FIG. 1a show that the stock formulation is stable with no insolublematerial or precipitate formed when maintained at room temperature forat least up to 60 days (during the 60 day length of this particularstudy, the formulation remained completely stable and precipitate free).In FIG. 1b is shown studies demonstrating that when the stockformulation is diluted with D5W, it maintained essentially 100%stability during the time frame studied (up to 15 hours at 1 mg/ml, andup to 25 hours at 0.5 mg/ml). Hydrophobic agents dissolved in our novelsolvent vehicles are suitable for prolonged (12± hours) infusion time,yet the stability still leaves a margin of time for convenient handlingin the pharmacy and on the medical floor prior to patientadministration. For example, if a planned clinical (Bu) treatment doseprescribes 1-5 mg/kg body weight, it will be desirable to arrive at aconcentration of 1-2 mg/mL. A stock formulation of 5 to 10 mg/mL of drug(Bu) in VE-acetone/PEG could be easily and conveniently diluted in D5Wor another aqueous infusion fluid to achieve the appropriate finaluse-concentration in a suitable administration volume. The cliniciancould then elect to infuse the drug over either short or prolonged timeperiods without having to exchange bags of infuscate that might beneeded if the formulation were physically unstable or subject to rapidchemical degradation. Finally, the clinician would be less dependent onwhether the routine pharmacy service hours permit a certain drugadministration schedule.

Enhanced Solubility in Physiologically Acceptable Solvents

The solubility of Bu was determined in several individual vehicles.Briefly, a known amount of Bu, formulated as a powder (Sigma, St Louis,Mo.), was equilibrated in the respective solvent at RT over 1-4 hours.An aliquot was removed, filtered, and further diluted in the HPLC mobilephase (or acetonitrile) before HPLC to determine solubility at apredetermined time. Since Bu is virtually insoluble in, and also israpidly hydrolyzed in (the presence of) water, the inventors insteadexamined non-aqueous organic solvent(s), and acetone was ultimatelyselected as a preferred first solvent. The Bu was dissolved at 10 mg/mL(maximum solubility of about 16 mg/mL, see Table 1), and this primaryBu-acetone composition was subsequently mixed with PEG (1:2, v/v). Thissecondary composition was further examined relative to an estimate ofhow to arrive at a (stable) stock formulation that would be useful in aclinical situation. The assumption was made, that intermittent“high-dose” administration or a prolonged infusion would be thepreferred modes of administration, i.e., choosing an infusion schedulethat would require the solvent vehicle to stably dissolve aconcentration of approximately 5-10 mg/mL. Since it was readilyrecognized that an organic solvent, such as acetone or chloroform, mightbe unsafe to routinely administer systemically in a human or domesticanimal, the inventors investigated removal of the organic solvent(acetone) by vacuum extraction.

TABLE 1 Solubility of Busulfan in various solvents at room tempaeratureSolvent Solubility (mg/mL) Acetic acid 3.8 HCl 6N 0.7 Ethanol 0.3Propylene glycol 0.2 DMA 49.2 DMSO 77.9 Acetone 16.4 (in literature ~25)PEG (−400) 0.2 PEG:water 1:1 0.25 PEG:water 1:2 0.19 Water Negligible(The Merck Index)

The inventors hypothesized that vacuum-assisted extraction of theprimary organic solvent at RT could create a microenvironment that wouldfacilitate a close electrostatic attraction between the hydrophobicdrug, Bu, and the amphiphilic polymer PEG, such that precipitation ofthe pharmaceutically active agent would not occur during the gradualremoval of the primary organic solvent. The inventors furtherhypothesized, that this electrostatic binding would be sufficientlystrong to allow the addition of a secondary aqueous diluent withoutimminent physical precipitation or chemical degradation of thepharmaceutically-active agent. Hence, the primary composite formulation(Bu in acetone and PEG) was subjected to vacuum extraction of theacetone at RT to create the clinical-stock formulation (i.e.Bu/VE-acetone/PEG), that would be miscible with the secondary aqueousdiluent, such as NS, 5% or 10% dextrose in water (D5W and D10W,respectively), or soybean lipid emulsion (Liposyn™, or Intralipid™,Pharmacia, Peapack, N.J.). This clinical-stock formulation was thendiluted with the secondary aqueous diluent to yield a stable clinicaluse-formulation. The desired range of Bu-concentrations in this finaluse-composition is 0.5-2 mg/mL, or more preferably 1-2 mg/mL, as Bucould then be infused parenterally without concern for fluid overload orother side effects related to the total volume infused.

Ultimately, a solvent vehicle composed of an organic solvent alone (forBu and Posaconazole) or an organic solvent mixed with a small amount ofan acid or an alcohol, such as benzylalcohol, to facilitate protonationof functional groups in the pharmaceutically active agent (e.g.itraconazole or posaconazole), to change the drug's polarity andtemporarily increase its solubility in the volatile organic solvent, andthereby increase the electrostatic attraction between thepharmaceutically-active agent and the secondary amphiphilic polymericagent, PEG, when the latter is added. The subsequent vacuum-assistedextraction of the primary organic solvent, e.g. acetone, which can beaccomplished at ambient temperature, will sterically optimize theconditions for electrostatic attraction and prevent precipitation of thepharmaceutically-active agent.

The primary organic solvent vehicle, acetone, allowed complete Busolubilization at concentrations of at least 15 mg/mL, and after mixingwith PEG and extraction of the acetone, Bu is stably bound in PEG aloneat a concentration of approximately 5-10 mg/mL. Subsequently, theinventors documented that Bu remained stable in solution in theBu/VE-acetone/PEG stock formulation for at least 60 days at RT (FIG. 1).Once reconstituted with D5W the final clinical use-formulation is stablefor at least 15 hours at a concentration of 0.5 to 2 mg/mL at RT.

HPLC Analysis.

Busulfan Derivatization

HPLC assay provides an accurate and sensitive detection system for lowconcentrations of Bu in solution, both protein-free mixtures andprotein-containing fluids (i.e., blood plasma), utilizing fluorescencedetection in the UV spectrum. Unfortunately, Bu is a small moleculewhich does not contain a chromophore. Therefore it was derivatized withdiethyldithiocarbamate (DDTC), which subsequently allows liquidchromatographic separation and detection in the UV-spectrum (Bhagwatwaret al., 1996; Andersson et al., 2000; Madden et al., 2007; Chow et al.,1997). The HPLC procedure was carried out as follows: Bu-containingsolution (500 μL) was mixed with an equal amount (500 μL) DDTC stocksolution (1.17 M in water). The mixture was vortexed for 30 sec androtated for 5 min on a Tube Rotator (Barnstead International, Dubuque,Iowa). The derivatized Bu was extracted from the reaction mixture with 2mL ethyl acetate, followed by centrifugation at 1000×g for 10 min(Thermo Electron Corporation, Waltham, Mass.). A 500 μL-aliquot of theethyl acetate layer was withdrawn and evaporated to dryness under avacuum evaporator (Scimetrics Inc., Houston, Tex.). The residue wasreconstituted by mixing on a vortex machine for 1 min with 250 μL of themobile phase and subjected to HPLC.

Chemicals

Busulfan (Bu), Sodium Diethyldithiocarbamate (DDTC) and Itraconazolewere obtained from Sigma (St. Louis, Mo.). Polyethylene glycol 400 (PEG400), ethyl acetate, acetonitrile and tetrahydrofuran were purchasedfrom Fisher Scientific (Pittsburgh, Pa.). All chemicals were HPLC gradeunless otherwise indicated. Posaconazole was extracted and purified fromthe commercially available clinical-use formulation (Noxafil™ suspensionfor oral use) in the medicinal chemistry core facility of the Departmentof Experimental Therapeutics at the University of Texas MD AndersonCancer Center.

HPLC assay

Busulfan

The HPLC system included: an analytical column (Nova-pak C18 with 4-μmbeads; 150 mm×3.9 mm; Waters Corporation, Milford, Mass.), anautosampler (Waters model 717 plus autosampler, a pump (Waters model600E system controller) set to deliver 1.2 mL/min and an UV detector(model Waters™ 486 Tunable Absorbance detector) set at 254 nm. Theisocratic mobile phase was a mixture of acetonitrile, tetrahydrofuran,and water at a ratio of 11:4:5. A volume of 30 μL was injected into HPLCfor quantitation of Bu. The HPLC assay was linear within a Buconcentration range of 0.1-20 μg/mL. The mobile phase flow rate was 1.2mL/min for Bu and 1.0 mL/min for Itra and Posa. The analytic system wasbased on previously established extraction and HPLC experience withbusulfan (Bhagwatwar et al., 1996; Andersson et al., 2002; Madden etal., 2007; Martin and Matzke, 1982; Chow et al., 1997).

Examples of authentic Bu chromatograms are shown in FIG. 3, showingchromatograms from the stability studies.

The HPLC conditions are described in Example 1. In these panels the druganalyzed was from the stability study, where Bu was dissolved in theprototype VE-acetone/PEG formulation and further diluted to 0.5 mg/mLand 1 mg/mL, respectively, using D5W as the final solvent. The HPLCretention time under the above conditions utilizing the Nova-Pak C18column was about 2.5-3 min. The assay was linear from at least 0.1 μg/mLto more than 20 μg/mL in protein-free solutions, i.e., for the solventsystem utilized in the formulation-feasibility and -stability studies(FIG. 2, Table 2). FIG. 2 is the Standard curve of Bu concentration vs.area under the curve (AUC) obtained in the HPLC assay used in thestability studies. The x-axis shows concentration in μg/mL, and they-axis shows the AUC. Analogous standard curves were prepared for thepharmacology studies in mice as appropriate.

TABLE 2 Busulfan Standard Curve - HPLC Concentration RT AUC average 20ug/mL 2.623 2086599 2.621 2132448 2047685 2.623 1924008 10 ug/mL 2.6231058978 1040774 2.625 1089246 2.625 974099 5 ug/mL 2.621 521298 5436352.619 558881 2.619 550726 2.5 ug/mL 2.613 272710 283674 2.584 3038962.584 274417 1.25 ug/mL 2.584 157211 162242 2.588 168973 2.591 1605420.625 ug/mL 92934 92649 92649 92690 92323

This HPLC assay consistently yielded high recovery and accuracy and aninitially lower sensitivity limit of about 1 μg/mL, but by fine-tuningthe technique as well as increasing the volume injected in the HPLCbeyond the initially used 30 μL, the lower sensitivity limit could bereproducibly improved to a consistent lower quantitative limit of about25 ng/mL, and a lowest limit of absolute detection of about 5-10 ng/mL(Bhagwatwar et al., 1966; Chow et al., 1997; Andersson et al., 2000;Madden et al., 2007). This HPLC technique was standardized and used forall the stability studies without any further modifications. For theplasma pharmacology study, the appearance of endogenous plasmaprotein-derived peaks in the chromatogram necessitated the addition ofan extraction/purification step; this was accomplished by precipitationof the proteins by adding two volumes of acetonitrile followed bycentrifugation and analysis as described.

HPLC Assay

Azole Compounds

The HPLC system was modified from Woestenborghs et al. (1987), andfurther modified for Posa (Notheis et al., 2006; Courtney et al., 2003;Jang et al., 2010). It used the same basic equipment set-up as describedabove for analyzing busulfan; thus, the pump was set to a flow rate of1.0 mL/min. The detection of respective azole was set at 261 nm for bothItra and Posa. The isocratic mobile phase was a mixture of 60%acetonitrile in H₂O plus 0.05% diethylamine. A standardized volume of 10μL was injected into HPLC column for quantitation of the respectiveazoles. Briefly, this summarizes the parameters used for the HPLCanalysis.

The retention time for Itra was 4.7-5.5 min and 2.5-3.0 min for Posa. Asexpected it varied somewhat with respect to which particular azolecompound was assayed. HPLC assay provides an accurate and sensitivedetection system for low concentrations of Itra (azole compounds) insolution, both protein-free mixtures and protein-containing fluids (suchas clinically obtained samples, e.g. blood plasma), utilizingfluorescence detection in the UV spectrum. For the detection awavelength of 261 was chosen, based on the inherent absorption andemission maxima of the azole molecules. This was varied as to whichparticular azole analog is examined, both the two prototype agents usedhere, Itra and Posa can be reliably detected at 261 nm.

All chemicals were HPLC grade unless otherwise indicated. Our analyticalsystem was based on previously established HPLC methodology for Itra(Woestenborghs et al., 1987).

To avoid analytical interference from endogenous plasma proteins in thechromatogram when assaying Itra and Posa in plasma samples, anextraction/purification step utilizing precipitation of protein materialwith acetonitrile was performed. Briefly, plasma proteins wereprecipitated by adding acetonitrile to a final volume ratioplasma:acetonitrile of 1:2. The mixture was vortexed for 30 seconds andcentrifuged for 5 min. at 14,000 rpm in an Eppendorff microcentrifuge.The deproteinated supernatant, containing Itra, was injected into theHPLC to determine the drug concentration.

Examples of authentic Itra chromatograms from the HPLC assay are shownin FIGS. 12 and 13. FIG. 12 depicts chromatograms obtained from the HPLCassay in the (protein-free) stability studies. The injected samplevolume was 10 μL. The HPLC conditions were as described above. In thesepanels the drug analyzed was from the stability study, where Itra wasdissolved in the prototype solvent vehicles (a), and further dilutedusing D5W as the final diluent (b). The HPLC retention time under theabove conditions utilizing the C18 Nova-Pak column was 4.7-5.5 min. Theassay was linear from 0.1 μg/mL to 100 μg/mL in protein-free solutions,i.e. the various solvent systems utilized in the formulation-feasibilityand -stability studies.

FIG. 14 depicts chromatograms from the plasma assay of Itra and Posa inthe pharmacology study. The data are below in Table 3.

TABLE 3 Clearance of a) Itraconazole and b) Posaconazole after injectionof 5 mg/kg IV in Swiss Webster mice. ug/mL mean SD a) Itraconazole Time,Min 10 3.59 3.23 0.76 2.13 3.34 3.86 30 1.31 1.62 0.44 1.93 60 0.77 0.77120 0.64 0.71 0.09 0.77 b) Posaconazole Time, hours 0.2 3.43 3.68 1.341.88 4.59 4.82 2.0 4.99 4.77 0.38 4.85 5.03 4.21 7.0 2.61 3.23 0.88 3.8624 0.03 0.35 0.28 0.56 0.47 30 0.14 0.16 0.13 0.29 0.04

This HPLC assay consistently yielded high recovery and accuracy and alower sensitivity limit of about 10-20 ng/mL, sufficient for the plannedexperiments. This HPLC technique was standardized and used for allstability studies without additional modifications, except asnecessitated by assaying the different azole-analogs.

Azoles parenteral formulations—The azole antibiotics Itra and Posa werealso formulated for parenteral infusion with VE-acetone/PEG/D5W, and thestability in VE-acetone/PEG at 4 mg/mL is displayed in FIG. 9. Whensubsequently diluted in D5W to a final concentration of 2 mg/mL theyremained stable for at least 15 hours at RT (FIG. 10). These novel azoleformulations retained full antifungal activity against selected strainsof Aspergillus and Mucor spp. (FIG. 11 and Table 4).

TABLE 4 Two common mold pathogens under standard conditions read-out at48 hr. Aspersillus-ATCC 90906 Mucorales (Rhizopus lab strain) Drug MIC¹Drug MIC Itra/S² 0.12 ug/ml Itra/S  0.3 ug/ml Itra/D³ 0.12 ug/ml Itra/D 0.6 ug/ml Posa/S⁴ 0.03 ug/ml Posa/S 0.06 ug/ml Posa/D⁵ 0.03 ug/mlPosa/D 0.12 ug/ml ¹MIC = minimum inhibitory concentration, ²Itra/S =itraconazole in solvent system S, ³Itra/D = itraconazole in DMSOcontrol. ⁴Posa/S = posaconazole in solvent system S, ⁵Posa/D =posazonazole in DMSO as a positive control solvent. Range of drugconcentrations tested 32 ug/ml to 0.03 ug/ml. Note: Drug is prepared ineach solvent system at the same initial concentration then diluted withmedia 1:50 to yield the concentration in the first well. All drugs arethen further diluted by 2 fold serial dilution in media through well 11.Well twelve is left as a drug free negative growth control.

Additional Mold Species were tested similarly as in Table 4. TheAspergillus strain tested is new Aspergillus fumigatus [LAB-A frompatient AF 040 511]. The Zygomycete tested is new Rhizomucor sp. [Lab-Zis derived from patient RM 041511]. The range of drug concentrationstested was 150, 25, 12, 6, 3, 1.5, 0.75, 0.38, 0.19, 0.09, 0.04, and 0(control) μg/mL. The susceptibility test was set-up using the standardmethod for filamentous fungi (CLSI, M38A standard). YeastOne media, TrekDiagnostics and Lot 152274SA—exp 2011 August were used. Aspergillusfumigatus LAB-A inoculum was made 82% transmission in Saline. Zygomycete(Lab Z) inoculum was made 80% transmission in Saline. Itra was suppliedat 1.5 mg/ml in vehicle. Posa was supplied at 2.0 mg/ml in vehicle. TheZygomycete was incubated at 33° C. for the first 24 hr to get growthestablished. The MIC results at 48 hrs were: Aspergillus LAB-A at 48 hrshowed an MIC of 0.09 μg/ml for Itra in novel solvent; Aspergillus LAB-Aat 48 hr showed an MIC of 0.04 μg/ml for Posa in novel solvent;Zygomycete LAB-Z at 48 hr showed an MIC of 0.2 μg/ml for Itra in novelsolvent; Zygomycete LAB-Z at 48 hr showed an MIC of 0.04 μg/ml for Posain novel solvent. Results showed that at 48 hr all growth controls werepositive and equal as expected. There were no discrepant wells among 4replicates at each drug concentration.

Example 2—Demonstration of In Vitro Solubility, Stability and OtherProperties of One of the Novel Formulations

In this example, stable Bu- and azole-(Posaconazole; Posa) formulationsthat would be suitable for human administration were evaluated. Thechemical and physical stability of Bu and Posa in (a) composite solventvehicle(s) were established. Further, the solubility of Bu and Posa inthese composite solvent vehicles was established, using NS or D5W±20%PEG as the final diluent. This example also investigated the in vitrocytotoxic and antifungal properties of the respective formulations,including their hemolytic potential, cytotoxic/antifungal activityagainst human malignant cell lines and mold isolates, to establish thatthe solvent system is appropriate for parenteral administration astherapy for malignant or autoimmune diseases as well as againstfungal/mold infections in humans and other mammals without loss of thetherapeutic properties of the respective class of agents.

Solubility Studies

Bu and Posa were dissolved in various solvents at room temperature (RT)for 60 minutes, and centrifuged at >14,000 rpm for 1 min. Thesupernatant was then analyzed using HPLC to determine the maximumBu/Posaconazole solubility. The Bu solubility differed markedly betweendifferent primary solvents (Table 1). A high equilibrium solubility(more than 15 mg/mL) was reached using DMA, DMSO, and acetone. In DMSO,Bu rapidly started degrading, confirming our concern that the sulfurgroup of DMSO would be reacting with Bu upon extended exposure. Anorganic solvent (e.g. acetone, or chloroform or DMA) was insteadidentified as a preferred primary solvent vehicle. Because of thetoxicity concerns about using chloroform and/or DMA as the primarysolvent (Dwivedi, 2002; VICH Steering Committee, 2010; The Food and DrugAdministration, 2010; The Office of Environmental Health HazardAssessment, 2010), the inventors continued the investigations withacetone as the preferred primary solvent. Busulfan could be stablydissolved (for at least 7 days at RT) in acetone and DMA, but oncedissolved, the Bu-acetone mixture could not be diluted with an aqueousinfusion fluid to allow parenteral administration without immediateprecipitation. In a second step the inventors therefore utilized amodified cosolvency approach (Spiegel and Noseworthy, 1963; Yalkowsky etal., 1981).

The inventors hypothesized, that since Bu is verylipophilic/hydrophobic, the combination of acetone as a primary organicsolvent combined with a secondary hydrophobic/amphiphilic carrier wouldsubject Bu to electrostatic attraction from the secondary polymericcarrier compound (e.g. PEG). The inventors further argued, that bygently removing the primary (volatile) organic solvent the inventorswould create a micro-environment that sterically allows graduallyincreasing electrostatic attraction/binding of solubilized Bu in acomplex with the carrier-molecule (e.g. PEG), and further, that thiscomplex would be sufficiently stable to allow the drug-complex to bediluted in an aqueous infusion fluid without immediate chemicaldegradation or physical precipitation of the pharmaceutically activeagent (in this example: Bu or Posa). The removal of acetone would befacilitated by adding a vacuum-extraction step that is facilitated bythe high volatility/low boiling point of acetone. The inventors furtherhypothesized that since PEG is readily water-soluble, its amphiphiliicproperties would make it a suitable “carrier substance”. The resultingdrug/VE-acetone/PEG complex could then be diluted in water or an aqueousinfusion fluid such as D5W or D10W or normal saline (NS) withoutprecipitation, allowing its application in medical use throughparenteral administration. The added benefit of the VE-acetone approachwould be that the inventors could minimize the recipient's ultimateexposure to the primary organic solvent vehicle(s).

Stability Studies

It was initially important to describe short-term stability of theBu/acetone/PEG mixture. This was necessary to determine if thevacuum-extraction step could be utilized without undue chemicaldegradation of Bu. The Bu (5 mg/mL) in acetone/PEG400 (1:2, v/v) wasincubated at room temperature and quantified by HPLC after 0, 1, 2, 4, 8and 24 hours. Second, the inventors confirmed that Bu would be stable inthe VE-acetone/PEG/Bu complex. Therefore, to determine long-termstability Bu was dissolved in acetone (10 mg/mL), then mixed with PEG400 (ratio 1:2, v/v). The acetone was extracted from the mixture undervacuum at room temperature (RT). The VE-acetone/PEG400/Bu was storedeither at room temperature or 37° C. for 2 months in sealed tubes.Finally, Bu (1 mg/mL) in the final use formulation of VE-acetone/PEG400/D5W (0:40:60, v/v/v), was incubated at room temperature and analyzedby HPLC at 0, 1, 2, 4, 8 and 15 hours to determine short-term stability.Triplicate samples were quantitatively analyzed with HPLC at all timepoints after appropriate dilution of the samples in the stabilitystudies.

PEG-400, PG, NS, D5W, and 20% soybean lipid emulsion (Intralipid™) didnot yield any significant concentrations of solubilized drug (<0.2 mg/mLafter 60 min at RT). The latter were therefore not considered forfurther study as primary solvents.

Stability of the Various Bu Formulations

The physical and chemical stability of the various intended parenteralformulations were studied as follows, using the Bu/VE-acetone/PEGformulation as an example, and using D5W as the prototype final diluent.

Bu was dissolved at a concentration of about 10 mg/mL in acetone only orin VE-acetone/PEG (prototype stock solvent vehicle) and incubated at RT(22° C.) and at 37° C. The resulting Bu concentration was measured byHPLC in samples taken immediately after solubilization, then every fewhours for 8 hours, and then at gradually increasing time intervals forup to 60 days.

In summary, the stability of Bu in the favored prototype solvent system(Bu/VE-acetone/PEG vehicle) was excellent: at 60 days ≥95% of Bu wasstill intact at RT, assayed by HPLC. From the available data it wasdeduced, that Bu and Posa in the described final solvent vehicle(s)is/are sufficiently stable for routine clinical use.

Hemolysis Studies In Vitro

The hemolytic potential of the final use-formulation should also beminimal, since the terminal use formulation/infusate contain Bu and PEGas the only significant solutes to be infused, i.e. this is basicallyD5W with a moderate amount of PEG (40%, v/v) as its main additive. Theprocedure of Parthasarathy et al. was used to examine the hemolyticpotential of a few selected formulations, and the LD₅₀ values of themost optimal formulation was constructed as previously described(Parthasarathy et al., 1994). Briefly, heparinized blood was mixed withan equal volume of Alsever's solution (Alsever and Ainslie, 1941). Thismixture was washed twice in PBS, and a 10% (volume per volume, v/v)erythrocyte/PBS solution was then prepared and mixed with increasingamounts of PEG (essentially the VE-acetone/PEG prototype vehicle) withor without addition of D5W and with or without Bu. The resultingmixtures were incubated for 4 hours at 37° C. At the end of theincubation, the cells were pelleted at 10,000×g in an Eppendorfmicro-centrifuge, and after washing twice in NS, the pellet wasresuspended and lysed using distilled water. The release of hemoglobinin the supernatant (i.e., hemolysis) was determinedspectrophotometrically at a wavelength of 550 nm. Maximum lysis wasmeasured against a reference erythrocyte solution that had been lysed byhypotonic shock. The hemolytic potential of the vehicle and the completefinal use-formulation (Bu/VE-acetone/PEG/D5W) was evaluated as described(Parthasarathy et al., 1984). The results were plotted as the fractionof intact erythrocytes versus concentration (total volume percent) ofthe solvent vehicle. The total volume percent was defined as the volumepercent of the solvent system in the mixture after addition of theerythrocyte suspension. This was done to simulate the dilution of thedrug formulation in the blood stream after parenteral administration.Intact, healthy erythrocytes were defined as those capable of retainingtheir hemoglobin intracellularly after mixture with the solvent vehiclewith or without Bu.

The final use-formulation showed a negligible capability for inducinghemolysis when the complete use-vehicle with or without Bu was used. TheBu-dependent lysis barely exceeded background levels, also for drugconcentrations of 100 μg/mL or more, which is many times higher than thehighest expected concentration in a clinical situation with once dailyBu-administration as a three-hour infusion (expected peak-concentrationsof less than 5 μg/mL) (Russell et al., 2002; De Lima et al., 2004;Madden et al., 2007). The Bu-specific hemolysis was highly reproduciblebetween different assays. The data derived from repeated experimentswith the complete final use-solvent vehicle±Bu are summarized in FIG. 4,which graphically illustrates the (lack of) hemolytic potential of theuse formulation of VE-acetone/PEG/D5W with Bu, and the same formulation(“solvent” alone) without Bu, as indicated in the figure. The solventalone curve also demonstrates the exceedingly low hemolytic potentialwhen the solvent is mixed with alternative agents including, but notlimited to, azole compounds. The x-axis shows the concentration inμg/ml. The y-axis shows the percent hemolysis. Finally, there was nosign of hemolysis after injection of 10 mg/kg in vivo, when urinesamples were evaluated up to 4 hours in parallel with obtaining bloodsamples for plasma concentration assays in the mouse experiments.

In conclusion, the Bu/VE-acetone/PEG/D5W complete infusion fluid hadvery low hemolytic potential and should be completely safe for humanparenteral (intravascular as well as intrathecal, or intracavitary)administration.

In Vitro Cytotoxicity of Busulfan.

The cytotoxic potential of the selected preferred solvent systems withand without Bu was determined against the human myeloid leukemia celllines KBM-3 and KBM-7/B5, as well as the OCI-AML3 (Andersson et al.,1993; Andersson et al., 1987; Andersson et al., 1995; Wang et al.,1991). Briefly, continuously growing cells were exposed to drug, andthen pelleted, after which they were resuspended in complete cellculture medium at 2×10⁵/mL and aliquoted (20,000/well) in 96-wellplates, incubated at 37° C. for 4 days, and analyzed by the MTT assay(Mosmann, 1983). Graphical analyses including calculations of IC₅₀ andcharting of survival were done using Prism 4 (GraphPad Software, Inc.,San Diego, Calif.). Cells exposed in parallel to solvent system andmedium alone served as negative controls, and cells exposed to DMA(final concentration 0.08% v/v) served as a positive control for acomparison of cytotoxic action with the DMA-Bu formulation.

The examined solvent system VE-acetone/PEG did not exhibit anydetectable toxicity in the concentrations achieved at the highest testedBu concentrations against any of the cell lines used in theseexperiments (negative control, data not shown). When the finalBu-use-formulation was added in increasing concentrations to the cells,concentration-dependent cytotoxicity was apparent (FIG. 5). Busulfanretained full cytotoxic activity in the use-formulation when comparedwith cells exposed in parallel to Bu dissolved in DMSO (positivecontrol). Interestingly, in these experiments the DMA-Bu formulationexerted significantly higher cytotoxic activity (FIG. 6); this activitywas higher than could be accounted for by additive cytotoxicity andindicates pronounced synergy between Bu and DMA in all examined celllines (Chou and Talalay, 1984). This observation triggered oursubsequent examination of whether the DMA by itself exerted significantand measurable cytotoxic activity. The DMA-solvent was highly toxicagainst both the AML cell lines KBM-3 and OCI-AML3, and it also hadsignificant cytotoxic activity against the CML line KBM-7 and itsBu-resistant subline B5/Bu250-6, FIG. 5(c). It is conceivable, that such(unpredictable) DMA-related toxicity could in a significant waycontribute to the clinical risk of receiving the DMA-Bu formulation,since normal organ-effects of DMA are likely to parallel what is seen inthe cell lines. This supposition is further supported by both theoccupational hazards literature and by the available toxicity data fromthe evaluation of DMA as an anti-cancer compound (Choi et al., 2001;Weiss et al., 1962a; Weiss et al., 1962b), as well as by theinvestigation of DMA-related fetal toxicity in rodents and logomorphs(Malley et al., 1995; Kennedy, 1986; Klimisch and Hellwig, 2000; Okudaet al., 2006; Valentine et al., 1997; Kennedy, 2001). Finally, aprevious investigation of DMA-Bu in vitro indicated synergisticcytoxicity between DMA and Bu in human leukemia cells (Sadeghi et al.,1999).

In Vitro antifungal activity of Itraconazole and Posaconazole.

The antimicrobial/antifungal potential of selected solvent systems withand without Itra was determined against several isolates of both yeastand different mold species. The findings confirm that the Itra, and Posaretain antifungal activity (Table 4). The variant solvent systems are inthemselves without any effect on mold- and yeast-proliferation (FIG.11).

Yeasts

Tested drug dilution range 38 μg/mL to 0.03 μg/mL

Candida cruzei (ATCC strain 6258) Candida parapsilosis Drug MIC Drug MICItra-s 0.07 Itra-s 0.03 Itra* 0.15 Itra* 0.07 Itra* is a control lot ofItra dissolved in DMSO as a positive control

Itra-s is the Itra dissolved in the new formulation system.

Growth controls (negative controls, fungae grown in medium only)displayed excellent growth. Candida growth in medium with solventvehicle without drug also displayed excellent growth.

B. Molds

Two hyaline molds were tested with a standard read out at 48 hrs:

Tested drug range: 75 μg/mL to 0.07 μg/mL

Aspergillus fumigatus Aspergillus fumigatus (ATCC strain 90906)(Clinical Lab isolate) Drug MIC Drug MIC Itra 1.2 Itra 0.6 Itra* 0.6Itra* 0.3 For description of Itra-s and Itra*, see above.

Extended Mold Testing

To further determine the antifungal activity of the compounds in the newformulation systems the inventors investigated the efficacy of thevarious agents against additional strains of mucor and Aspergillus (TheRhizomucor was a clinical isolate from a patient isolate) and theAspergillus fumigatus (ATCC strain 90906) used was as previouslydescribed. Again, the susceptibility tests were set-up using thestandardized test method (CLSI M38A). The used drugs were provided inthe described final use-formulation VE-acetone/PEG/D5W. All drugs werediluted in RPMI-Mops medium: YeastOne, Sensititer (Lot 151416SA,expiration date in 2011).

As before two different molds were tested with a standard read out at 48hr:

Drug dilution range 75 μg/mL to 0.07 μg/mL.

Aspergillus fumigatus Zygomycete (Clinical Lab (ATCC strain 90906)isolate, MDACC) Drug MIC Drug MIC Itra 1.2 Itra 2.5 Itra* 0.3 Itra* 2.5All mold growth controls, including controls with solvent vehicle(s)without added azole drug, grew without inhibition as described before.For description of Itra*, see above.

Briefly, susceptibility tests were set-up using a standardizedmethodology (CLSI M38A). Drugs were diluted into RPMI-Mops medium (YeastOne Broth (Sensititer, product Y3462, Trek Diagnostic Systems,Cleveland, Ohio) Sensititer Lot number 151416SA-expiration date 2011).Two different strains of yeast were tested, the standardizedevaluation/read out was performed at 24 hours after the start of eachculture. The tests were repeated twice and all MIC values (minimuminhibitory concentration) determined as an average of the twoexperiments.

Example 3—Quantitative Busulfan Analysis in Plasma and Pharmacology ofIV Bu

This example demonstrates that Bu and azole antifungal antibiotics in(a) preferred solvent vehicle(s) and mixed with blood plasma may berecovered as native/intact drug using quantitative extraction technologyand HPLC assay, and that the drugs remain stable in acytotoxic/fungistatic concentration range for several hours afteradministration. It further shows that the preliminary plasmapharmacokinetics after parenteral administration of a preferred Buformulation in mice conforms to what can be expected, based on thepublished pharmacology of oral and IV Bu (Slattery et al., 1997; Dix etal., 1996; Hassan et al., 2000; Hassan et al., 1989; Bhagwatwar et al.,1996; Andersson et al., 2000; Andersson et al., 2002; Russell et al.,2002; De Lima et al., 2004; Madden et al., 2007; Busulfex, 2009).

Quantitative Extraction of Busulfan in Plasma

One mL of human plasma was mixed with various amounts of thereformulated Bu (<3% of the final volume) to yield a drug concentrationrange from 1 to 10 μg/mL (the use-formulation Bu/VE-acetone/PEG/D5W wasmade of the prototype solvent vehicle and D5W, having a Bu concentration1.0 mg/mL). The drug was then extracted from the plasma samples andanalyzed by HPLC as described in Example 1. Briefly, 1 volume of bloodplasma was mixed with 1 volume of acetonitrile and after vortex-mixingthe samples were centrifuged to precipitate the plasma proteins thatwould otherwise produce interfering peaks in the chromatograms. Thesupernatant was then derivatized with DDTC and extracted with ethylacetate. After evaporation-reconstitution the Bu was assayed by HPLCusing fluorescence detection at 254 nm as described above. The Buretention time in this system was 2.5-3.0 min when using the Nova-Pakcolumn (see Example 1). The Bu recovery from plasma spiked at 10 μg/mLwas calculated to be approximately 90-100% (data on file). The assay waslinear in the interval from about 25 ng/mL to at least 20 μg/mL, with adetection limit of about 5-10 ng/mL when 100 μL was injected into thechromatograph. A standard curve was routinely prepared in the range from0.10 μg/mL to 10 μg/mL for the pharmacology experiment (not shown), anda good linear correlation (r=0.99) was obtained between the actualplasma Bu concentrations and the measured AUC values.

Parenteral Busulfan: Experimental Protocol in Mice.

Swiss Webster mice with a body weight of 25-30 g were used for the invivo pharmacology experiments (Harlan-Sprague-Dawley, Houston, Tex.).The animals were allowed a minimum of 3-4 days after arrival toacclimatize to the new environment and allowed free access to commercialfeed and hyperchlorinated, reverse-osmosis purified water prior to andduring the experimentation period. The animal-housing facilities meetthe requirements of the USDA, NIH, and DHHS.

Busulfan at a dose of 10 mg/kg BW was used as an appropriate andrepresentative single dose that could be administered as a slow IV bolusinjection over 3-4 min.

The Bu was formulated in the prototype VE-acetone/PEG vehicle to a stockconcentration of about 10 mg/mL and then diluted with D5W to 1 mg/mL and2 mg/mL (in repeated experiments) so the total dose of 10 mg/kg could beinjected in a volume of about 250 μL and 125 μL, respectively, in a tailvein. The Bu concentrations of the final use-formulation were confirmedby HPLC prior to administration.

No anticonvulsant premedication was used in the animals in theseexperiments to avoid the possible induction of microsomal liver enzymesthat could modify Bu metabolism. For the same reason the animals wereunanesthetized during the drug injection, only physically restrained.

Blood samples (0.5-1.0 mL) were procured through cardiac puncture underIsoflurane-induced general anesthesia. These samples were drawn inpreheparinized tubes at selected time points prior to the drug infusion(“blank”), and from 5 min to 4 hours after drug injection fordetermination of Bu concentrations. The blood was centrifuged at 1,000×gfor 10 min, and plasma was removed and stored at −80° C. until extractedand assayed by HPLC.

Busulfan in Plasma and IV Drug Pharmacology Results

Authentic chromatograms from (a), Bu-spiked plasma and (b) one plasmasample obtained from the current pharmacokinetic study are shown in FIG.7. FIG. 7 shows a plasma sample spiked with Bu in the new prototypeformulation to 10 μg/mL, and the figure also shows a chromatogram fromthe pharmacology study, where mice were injected with Bu at 10 mg/kg.The latter chromatogram was from a sample drawn 20 minutes after druginjection.

The resulting data illustrate that the novel Bu solvent vehicle(s)give(s) detectable, cytotoxic Bu plasma concentrations after injectionof 10 mg/kg BW. FIG. 8 is a graph showing the change in plasmaconcentration over time after a Bu injection of 10 mg/kg injected IVover approximately 3-4 min. The x-axis shows the time after injection inminutes. The y-axis shows the Bu concentration in μg/mL plasma, with anapparent terminal Bu half-life in the range of 2.5-3.5 hours. Allinjections were well tolerated, and no visible adverse effects wereencountered during the acute 4-hour observation period.

In summary, the data demonstrate that the novel pharmaceuticallyacceptable, stable formulations of Bu in the invention can be safelyutilized for parenteral administration. A preferred solvent vehicle isphysiologically compatible with intravascular administration and wasused to demonstrate that the injection of Bu in this solvent vehicle waswell accepted and had insignificant acute solvent system toxicity in amouse model. The IV injection of this formulation in mice at 10 mg/kg BWyielded Bu plasma concentrations that for several hours remained in thecytotoxic range, based on comparisons with our in vitro cytotoxicityexperiments and in comparison with data obtained from several earlierstudies using either oral Bu (Slattery et al., 1997; Dix et al., 1996;Hassan et al., 2000; Hassan et al., 1989; Vassal, 1994; Hassan et al.,1994), or DMA-Bu (Andersson et al., 2000; Andersson et al., 2002;Russell et al., 2002; De Lima et al., 2004; Madden et al., 2007).

The data obtained with the final use-formulation VE-acetone/PEG/D5Wconclusively prove that it is now feasible to introduce Bu forparenteral administration in clinical therapy of malignant andautoimmune diseases without the added toxicity of DMA used as anexcipient component of the solvent vehicle. This can be expected toresult in greatly improved safety of the Bu-based cytotoxic treatmentprogram. These results now finally also give a reasonable expectation ofinsignificant normal organ toxicity from the solvent vehicle. Inparticular, it is possible that the serious hepato-renal and centralnervous system toxicity that is still occasionally encountered with thecurrently available DMA-Bu formulation may be avoided with this newsolvent vehicle(s).

The novel solvent systems will significantly improve the clinical safetyof Bu-based therapy, and make it possible to optimize the use of thisimportant drug in the treatment of cancer and autoimmune disorders. Inparticular, embodiments of the invention may be utilized in conditioningof patients undergoing combination chemotherapy preceding HSCT.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention.

Abbreviations Used in this Application

AAALAC—Association for the Assessment and Accreditation of LaboratoryAnimal Care International

ALT—Alanine-Amino-Transferase.

Alsever's solution—Standardized solution of citric acid in SodiumChloride (Alsever and Ainslie, 1941)

AML—Acute myeloid leukemia.

AST—Aspartate-Amino-Transferase.

ATCC—American Tissue Culture Collection, Rockville, Md.

ATG—Antithymocyte globulin.

AUC—area under the curve, term used to denote the actual measured areaof a peak in a chromatogram, and also for the area under the plasmaconcentration vs. time curve over several hours after administration ofa drug to an animal or human being.

BSA—Body surface area.

Bu—1,4-butanediol dimethanesulfonate, Butane-1,4-diyl dimetanesulfonate;C₆H₁₄O₆S₂, Busulfan

BW—Body weight.

Clo—Clofarabine.

CLSI M38A standard—Clinical Laboratory Standards Institute M38A(Standardized Susceptibility testing for filamentous molds).

CML—Chronic myelogenous leukemia.

Cy—Cyclophosphamide.

D5W—Dextrose, 5% in water

D10W—Dextrose, 10% in water

DMA—N,N-Dimethylacetamide.

DMF—Dimethylformamide.

DMSO—Dimethylsulfoxide.

DNA—Deoxyribonucleic acid.

DHHS—Department of Health and Human Services.

EtOH—Ethanol.

FBS—Fetal bovine serum.

FDA—United States Food and Drug Administration.

Flu—Fludarabine.

GSH—Glutathione.

GST—Glutathione-S-Transferase.

HPLC—High-pressure liquid chromatography.

HSCT—Hemopoietic stem cell transplantation.

IMDM—Iscove's modified Dulbecco medium (GIBCO, Grand Island, New York,N.Y.).

Intralipid™—Brand name of an aqueous lipid emulsion made primarily fromsoybean oil and marketed for parenteral nutrition available from BaxterHealthcare, Inc. USA.

IV—Intravenous.

KBM-3—Human myeloid leukemia cell line designated KBM-3.

KBM-3/Bu2506—subline of the KBM-3 line resistant to busulfan.

KBM-7 and KBM-7/B5—Human myeloid leukemia cell line designated KBM-7,and a cloned subline thereof designated KBM-7/B5.

LD₅₀—The concentration or dose that results in 50% lethality ordestruction of a population.

Liposyn™—Brand name of an aqueous lipid emulsion made primarily fromsoybean oil and marketed for parenteral nutrition available from Abbott(Abbott Park, Ill.).

MDACC—University of Texas MD Anderson Cancer Center, Houston, Tex.

MDS—Myelodysplastic syndrome.

MeOH—Methanol.

MIC—term denoting the minimum inhibitory concentration of bacterial andfungal growth for an antibiotic when tested in a standardized in vitrosystem for assessment of sensitivity to said antibiotic.

MTT—3,[4,5-Dimethylthiazol-2-yl] 2,5-diphenyltetrazolium-bromide.

NCI—National Cancer Institute.

NH4-acetate—Ammonium acetate.

NIH—National Institute of Health.

NS—Normal saline (150 mM NaCl).

OCI-AML3—Human myeloid leukemia cell line.

PBS—Phosphate-buffered saline (Dulbecco's formulation, pH 7.4).

PEG, and PEG400—Polyethylene glycol(-400).

PG—Propylene glycol.

PTFE—Polytetrafluoroethylene (filters), Teflon™

RT—Room temperature (about 22° C.).

SDS—Sodium dodecylsulphate.

TBI—Total body irradiation.

TRM—Treatment-related mortality.

USDA—US Department of Agriculture.

UV—ultraviolet.

VE-acetone—see below

VE-acetone/PEG—PEG that after mixing with acetone subsequently has hadthe (volatile) acetone extracted/evaporated under vacuum, i.e.“Vacuum-Extracted, or Vacuum-Evaporated”.

VOD—Veno-occlusive disease.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention.

REFERENCES

The following references, to the extent that they provide exemplary,procedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 5,430,057-   U.S. Pat. No. 5,559,148-   Aggarwal et al., Biol. Blood Marrow Transplant., 12:770-777, 2006.-   Alsever and Ainslie, State Med. J., 41:126-135, 1941.-   Andersson et al., Biol. Blood Marrow Transplant., 14:672-684, 2008.-   Andersson et al., Biol. Blood Marrow Transplant., 8:145-154, 2002.-   Andersson et al., Bone Marrow Transplant., 6:548-554, 2000.-   Andersson et al., Cancer Genetics Cytogenet., 24:335-343, 1987.-   Andersson et al., Exp. Hematol., 20:361-367, 1992.-   Andersson et al., Leukemia, 9:2100-2108, 1995.-   Baddley et al., J. Clin. Microbiol., 47(10):3271-3275, 2009.-   Bearman, Blood, 85:3005-3020, 1995.-   Benet and Sheiner, In: Goodman and Gilman's The Pharmacological    Basis of Therapeutics, Goodman et al. (Eds.), 7^(th) Ed., MacMillan    Publishing Co. NY, P. 8, 1985.-   Bhagwatwar et al., Cancer Chemother. Pharmacol., 37:401-408, 1996.-   Blaise et al., Blood, 79:2578-2582, 1992.-   Boothe et al., Am. J. Vet. Res., 58(8):872-77, 1997.-   Bredeson et al., Biol. Blood Marrow Transplant., 14:993-1003, 2008.-   Busulfex, (IV Busulfan) for injection, package insert, Otsuka    America Pharmaceuticals, Inc., 2009.-   Campo et al., J. Infect. Dis., 60(5):331-337, 2010.-   Carrillo-Munoz et al., J. Antimicrobial. Chemoth., 55(3):317-9,    2005.-   Chae et al., Bone Marrow Transplant., 40:541-547, 2007.-   Chan et al., Bone Marrow Transplant., 29:963-965, 2002.-   Chen et al., Curr. Opin. Pharmacol., 10(5):522-530, 2010.-   Choi et al., Korean J. Occup. Environ. Med., 13:164-170, 2001.-   Chou et al., Adv. Enzyme Regul., 22:27-55, 1984.-   Chow et al., J. Chromatogr. B, 704:277-288, 1997.-   Ciurea et al., Biol. Blood Marrow Transplant., 15(5):523-536, 2009.-   Courtney et al., Antimicrob. Agents Chemother., 47:2788-2795, 2003.-   Courtney et al., Antimicrobial. Agents Chemo., 47(9):2788-2795,    2003.-   Davis et al., Am. J. Vet. Res., 66(10):1694-1701, 2005.-   De Lima et al., Blood, 104:857-864, 2004.-   Dean et al., Br. J. Haematol., 148:226-234, 2010.-   Deeg et al., Biol. Blood Marrow Transplant., 5:316-321, 1999.-   DeLeve et al., Semin. Liver Dis., 22:27-42, 2002.-   Devergie et al., Blood, 85:2263-2268, 1995.-   Dix et al., Bone Marrow Transplant., 17:225-230, 1996.-   Dodds-Ashley et al., Drugs of Today, 41(6):393-400, 2005.-   Dodds-Ashley, Pharmacotherapy, 30(8):842-854, 2010.-   Dutkiewicz and Hage, Proc. Am. Thorac. Soc., 7(3):204-209, 2010.-   Dwivedi, Pharmaceutical Technol. Europe, 42-46, 2002.-   Evans, Proc. Am. Thorac. Soc., 7(3):197-203, 2010.-   Evensen, et al., Thromb. Diath. Haemorrh., 19:570-577, 1968.-   Fortner et al., Am. J. Hosp. Pharm., 32:582-84, 1975.-   Glockner and Karthaus, Mycoses. 2010 (e-pub.)-   Greer, Baylor Univ. Med. Center Proc., 20:188-196, 2007.-   Grigg et al., Ann. Intern. Med., 111:1049-1050, 1989.-   Grochow et al., Cancer Chemother. Pharmacol., 25:55-61, 1989.-   Grochow, Semin. Oncol., 3:20 (Suppl 4):18-25, 1993.-   Groll and Walsh, Mycoses, 49 (Suppl 1):7-16, 2006.-   Haddow and Timmis, Lancet., 31:207-208, 1953.-   Hartman et al., Bone Marrow Transplant., 22:439-443, 1998.-   Hassan et al., Blood, 84:2144-2150, 1994.-   Hassan et al., Bone Marrow Transplant., 25:915-924, 2000.-   Hassan et al., Cancer Chemother. Pharmacol., 33:181-186, 1993.-   Hassan et al., Eur. J. Clin. Pharmacol., 36:525-530, 1989.-   Hempel et al., J. Clin. Oncol., 25:1772-1778, 2007.-   Hicheri et al., Expert Rev. Anti. Infect. Ther., 8(9):1049-1060,    2010.-   Hoffman et al., In: Hematology. Basic principles and practice,    Churchill Livingstone Inc., NY, Pa., 1991.-   Hsu et al., Infect. Dis. Clin. North Am., 24(3):557-577, 2010.-   Ito et al., Leuk. Lymphoma, 51(9):1623-1631, 2010.-   Jang et al., Clin. Pharmacol. Ther., 88(1):115-119, 2010.-   Jones et al., Transplantation, 44:778-783, 1987.-   Kashyap et al., Biol. Blood Marrow Transplant., 8:493-500, 2002.-   Kennedy, Crit. Rev. Toxicol., 31:139-222, 2001.-   Kennedy, Drug Chem. Toxicol., 9:147-170, 1986.-   Kim et al., Haematologica., 90:285-286, 2005.-   Kim et al., Mycoses., 54:e301-312, 2011.-   Klimisch and Hellwig, Human Experim. Toxicol., 19:676-683, 2000.-   Kobayashi et al., Bone Marrow Transplant., 21:217-220, 1998.-   Lee et al., Ann. Hematol., 84:321-330, 2005.-   Lehrnbecher et al., Eur. J. Clin. Microbiol. Infect. Dis.,    29:1043-1045, 2010.-   Lewis and Kontoyiannis, Med. Mycol., 47(Suppl 1):S271-281, 2009.-   Lortholary et al., Antimicrob. Agents Chemother., 54(10):4446-4450,    2010.-   Madden et al., Biol. Blood Marrow Transplant., 13:56-64, 2007.-   Malley et al., Fundam. Appl. Toxicol., 28:80-93, 1995.-   Marcus and Goldman, Lancet., 2:1463, 1984.-   Martell et al., Ann. Intern. Med., 106:173, 1987.-   Martin and Matzke, J. Clin. Pharm., 1:42-48, 1982.-   McDonald et al., Ann. Intern. Med., 118:255-267, 1993.-   McDonald et al., Blood, 101:2043-2048, 2003.-   Meloni et al., Ann. Oncol., 3:145-148, 1992.-   Meloni et al., Haematologica., 80:532-534, 1995.-   Mosmann, J. Immunol. Methods, 65:55-63, 1983.-   Notheis et al., Mycoses, 49(Suppl 1):37-41, 2006.-   Okuda et al., J. Occup. Health, 3:154-160, 2006.-   Pappas et al., Clin. Infect. Dis., 15; 50(8):1101-1111, 2010.-   Parthasarathy et al., Cancer Chemother. Pharmacol., 34:527-34, 1994.-   Person et al., Infect. Dis. Clin. North Am., 24(2):439-459, 2010.-   Peters et al., Cancer Res., 47:6402-6406, 1987.-   Russell et al., Biol. Blood Marrow Transplant., 8:468-477, 2002.-   Sadeghi et al., Proc. Amer. Assoc. Cancer Res., (Abstr), 1999.-   Santarone et al., Biol. Blood Marrow Transplant., 2011 (E-Pub-ahead    of print)-   Santos and Tutschka, J. Natl. Cancer Inst., 53:1781-1785, 1974.-   Santos et al., N. Engl. J. Med., 309:1347-1353, 1983.-   Santos, Bone Marrow Transplant., 4(Suppl 1):236-239, 1989.-   Shimoni et al., Leukemia, 20:322-328, 2006.-   Shimoni et al., Leukemia, 24:1050-1052, 2010.-   Singh et al., Transplantation, 81(3):320-326, 2006.-   Slattery et al., Blood, 89:3055-3060, 1997.-   Socie et al., Blood, 98:3569-3574, 2001.-   Spiegel and Noseworthy, J. Pharm. Sci., 52:917-927, 1963.-   Sureda et al., Ann. Intern. Med., 111:543-544, 1989.-   Thall et al., Bone Marrow Transplant., 33:1191-1199, 2004.-   The Food and Drug Administration, The Federal Register, Aug. 18,    2010.-   The Merck Index. Busulfan. p. 253, 2001.-   The Office of Environmental Health Hazard Assessment (OEHHA) within    the California Environmental Protection Agency, Chemical Listed    Effective May 21, 2010.-   Torres et al., Lancet Infect, Dis., 5(12):775-785, 2005.-   Tutschka et al., Blood, 70:1382-1388, 1987.-   Ullmann et al., N. Engl. J. Med., 356(4):335-347, 2007.-   Valentine et al., Inhalation Toxicol., 9:141-158, 1997.-   Van de Velde et al., Pharmacotherapy, 16(3):424-428, 1996.-   Vassal et al., Cancer Chemother. Pharmacol., 24:386-390, 1989.-   Vassal et al., Cancer Res., 50:6203-6207, 1990.-   Vassal, Anticancer Res., 14:2363-2370, 1994.-   Vehreschild et al., J. Antimicrob. Chemother., 65(7):1466-1471,    2010.-   VICH Steering Committee, VICH GL 18 (R)—Intl. Coop. Harmonisation of    Tech. Requirements for Registration of Veterinary Medicinal    Products. (Rev. 9), 2010.-   Walsh et al., Antimicrob. Agents Chemother., 54(10):4116-4123, 2010.-   Wang et al., Leukemia, 5:493-499, 1991.-   Weiss et al., Cancer Chemother. Rep., 16:477-485, 1962a.-   Weiss et al., Science, 136:151-152, 1962b.-   Willems et al., J. Clin. Pharm. Ther., 26(3):159-169, 2001.-   Wingard et al., Blood, 116(24):5111-5118, 2010.-   Winston et al., Biol. Blood Marrow Transplant., 2010 (e-pub).-   Woestenborghs et al., J. Chromatogr., 413:332-337, 1987.-   Yalkowsky and Roseman, In: Techniques of solubilization of drugs,    Yalkowsky (Ed.), Marcel Dekker Inc., NY, 91-134, 1981.-   Zhou et al., Clin. Pharmacol., 38(7):593-602, 1998.

What is claimed is:
 1. A homogeneous, non-aqueous pharmaceuticallyacceptable solution comprising busulfan solubilized in a non-aqueouspolyethylene glycol (PEG), the solution being pharmaceuticallyacceptable, homogeneous and free of non-polymeric organic solvents andnon-solubilized particles.
 2. The solution of claim 1, wherein said PEGis selected from the group consisting of PEG-100, PEG-200, PEG-300,PEG-400, PEG-600 and PEG-800.
 3. The solution of claim 1, wherein thesolution remains stable and free of non-solubilized lipophilicpharmaceutical particles for at least 40 days when stored at roomtemperature.
 4. The solution of claim 1, wherein the busulfan is presentin a concentration of about 2 to 10 mg/mL.
 5. The solution of claim 4,wherein the busulfan is present at a concentration of about 3 to 9mg/mL.
 6. The solution of claim 1, wherein the solution ispharmaceutically acceptable for parenteral administration.